Sampling site characteristics
Based on Table 1, there has been no statistically validated correlation between environmental variables. Soil pH generally decreases with increasing elevation, although Component B has the lowest pH (6.0). This phenomenon may be due to the fact that nutrients are more easily filtered and the microclimate is cooler. This observation is purely descriptive and lacks validation through inferential statistical analysis. The observed pH range (6.0-6.5) in the tropical highland region, where intense weathering and leaching due to high rainfall increase acidity, is ecologically significant because soil pH greatly controls nutrient availability, microbial activity, and organic matter decomposition. Moreover, the narrow variation in environmental temperature (25-26 °C) with a relatively uniform microclimate, although statement 14 explains that temperature can affect carbon and nitrogen dynamics in the soil, the narrow temperature range at this location limits its explanatory power regarding microbial distribution. Differences in wind direction have an impact due to exposure to sunlight and moisture retention, both of which are known as important factors in the heterogeneity of soil properties.

Table 1. Chemical Characteristics of Composite Soil at the Research Site: Coordinates, Elevation, Cardinal Direction, Elevation Difference (Elevation), Environmental Temperature, and Environmental pH

Description: e-T refers to ambient environmental temperature, and e-pH refers to environmental pH conditions

Analysis of soil nutrient content of biological education and research forest (HPPB), University of Andalas
The results of the macronutrient (N, P, K) content analysis at the three sample composite points revealed significant spatial variations in soil fertility status. The data showed a consistent pattern of decreasing nutrient concentrations from Composite A (T1 + T2) towards Composite C (T5 + T6). The entire study site showed serious nitrogen (N) deficiency, with levels ranging from 15-34 mg/kg (“Very Low” to “Low” category). This phenomenon is consistent with the typical characteristics of tropical rainforest soils, which have fast N mineralization rates but are susceptible to nutrient losses through intensive leaching processes.¹⁹ This condition is exacerbated by the high rainfall in tropical ecosystems that accelerates the leaching process, especially in soils with sandy textures.²⁰

Phosphorus (P) analysis revealed a more complex variability in fertility status, ranging from the “High” category in Composite A (38 ± 0.9 mg/kg) to “Moderate” in Composites B and C. The high P levels in Composite A are strongly suspected to be related to the optimal phosphate-solubilizing microbial activity at pH 6.5,²¹ as well as the accumulation of organic matter at the site. However, the 50% decrease in P levels in Composite C (19 ± 0.5 mg/kg) reflects the spatially limited availability of available P, a characteristic commonly found in old tropical soils.²² This uneven pattern of P distribution could potentially affect vegetation composition, where low-P adaptive species will tend to dominate in zones with lower P content.²³

Potassium (K) showed a similar downward trend to N, with Composite C having the lowest levels (42 ± 1.7 mg/kg) which is classified as “Very Low”. This low K availability can be explained by two main mechanisms: first, the high intensity of nutrient leaching in tropical rainforest ecosystems,²⁰ and second, the immobilization of K by the dominant 1:1 type clay minerals (kaolinite) in acidic soils.²⁴ These findings have important implications for ecosystem restoration management, where interventions through the addition of organic fertilizers or the implementation of agroforestry systems with N-fixing legumes are potential solutions.²⁵

Spatial variation of npk and ecological implications in tropical rainforests
Analysis of macronutrient content in the three composite soil samples (Table 2) revealed a clear trend of declining soil fertility from Composite A (T1 + T2) to Composite C (T5 + T6). Nitrogen (N) decreased from 34 mg/kg in Composite A, 24 mg/kg in Composite B, to 15 mg/kg in Composite C. Phosphorus (P) also exhibited a similar decreasing pattern, from 38 mg/kg in Composite A, 29 mg/kg in Composite B, to 19 mg/kg in Composite C. Potassium (K) declined from 88 mg/kg in Composite A, 65 mg/kg in Composite B, to 42 mg/kg in Composite C. This spatial variation appears to be associated with site characteristics, such as elevation and cardinal direction, which influence soil nutrient dynamics. The relatively low N concentrations, particularly in Composite C, are consistent with previous reports of low N retention in tropical soils due to a combination of high denitrification rates (>25 °C, +2.4 mg N/kg/day) at these temperatures (Table 1: 25-26 °C)²⁶ and low cation exchange capacity (CEC < 4 cmol(+)/kg) in coarse-textured soils.²⁷ The decrease in P content with elevation is also influenced by soil pH (6.0-6.5),²⁶ where microbial phosphatase activity is optimal at pH 6.3-6.8,²⁸ as well as by soil organic matter content. The decline in K reflects the dominance of kaolinite minerals, which generally have a low CEC (3-15 cmol(+)/kg).²⁹ Ecologically, the variation in N, P, and K is significant because it controls nutrient availability, microbial activity, and organic matter decomposition in tropical rainforest soils. The observed gradient of macronutrients provides insight into soil heterogeneity at the study sites and its implications for ecosystem productivity.

Table 2. Nutrient Composition (N, P, K) and Fertility Classification of Composite Soil Samples at HPPB, University of Andalas

Microbial Population Dynamics on Selective Media (CMC Agar and Tanat Agar)
Soil microbial populations in the HPPB Tropical Rainforest were analyzed based on their ability to degrade cellulitic (CMC Agar) and lignocellulolytic (Tanat Agar) can be seen in Table 3. The following data shows the average number of microbial colonies (×10⁴ CFU/g) along with the standard deviation at each sampling point.

Table 3. Microbial Population on CMC (Cellulytic) and Tanat Agar (Lignocellulolytic) selective media (×104 CFU/g)

Description: Values are presented as mean ± SD (×10⁴CFU/g). Different superscript letters (a-c) within the same column indicate significant differences at P < 0.05

Based on Table 3, information was obtained about significant differences in microbial growth characteristics between CMC and Tanat Agar media. In CMC media, the variability of cellulolytic microbial population showed a very high standard deviation value, especially in sample T3 + T4 (26.9 ± 157.5 ×10⁴ CFU/g), where the SD value exceeded the mean value. This phenomenon is consistent with the findings of Casey et al.³⁰ who reported that the natural heterogeneity of lignocellulosic substrates can cause significant variation in colony numbers. The high coefficient of variation (CV > 100%) in all CMC samples indicates the need for optimization of the isolation method, including the possibility of adding a longer acclimatization period or modifying the media composition.

In contrast, Tanat Agar medium showed better population stability, with SD of 0.0 at T1 + T2 and T5 + T6, indicating consistent growth of lignocellulolytic microbes. This finding is consistent with previous studies that reported the effectiveness of tannins as selective agents capable of suppressing the growth of non-target microorganisms.³¹ A marked difference was observed in the T5 + T6 sample, where the microbial population in Tanat Agar (50.0 × 10⁴ CFU/g) was significantly higher than in CMC Agar (8.4 × 10⁴ CFU/g). This may be attributed to site-specific adaptation of the microbial community to lignin degradation components, as previously reported regarding functional specialization of lignocellulolytic microbes.²⁹ The distribution of cellulolytic and lignocellulolytic microbes is shown in Figure 2.

The methodological implications of these findings inform the importance of a multisampling approach to overcome the heterogeneity of natural substrates, especially when using CMC media. Optimization of culture conditions, including the addition of pH buffers and modification of incubation time, may be necessary to improve the reproducibility of results. In addition, these findings underscore the need for complementary analyses such as enzyme activity assays and molecular identification to gain a more comprehensive understanding of microbial population dynamics.

The main limitations of this study include the relatively short incubation duration and the lack of molecular characterization of the isolates. For further research, it is recommended to apply a multi-omics approach that integrates metagenomic data, proteomics, and enzyme activity analysis to uncover the underlying mechanisms that control the population dynamics of lignocellulolytic microbes in different types of selective media.

The growth characteristics of microbial populations on selective media, as presented in Figure 3, show that Panel A illustrates cellulolytic microbial colonies on CMC Agar medium at a 10^-4 dilution, while Panel B shows lignocellulolytic microbial colonies on Tanat Agar medium at the same dilution; visual observation indicates differences in microbial composition and colony density between the two media, with CMC Agar (Panel A) displaying more prominent bacterial colonies, appearing as numerous small cream-colored colonies, although fungal growth, represented by larger filamentous colonies, is also present, whereas on Tanat Agar (Panel B), fungal colonies are clearly dominant, forming large white circular colonies, and bacterial colonies are relatively sparse, suggesting that Tanat Agar medium is more conducive to fungal growth, whereas CMC Agar medium favors bacterial proliferation, providing descriptive insights into the selective growth preferences of cellulolytic and lignocellulolytic microorganisms under the given experimental conditions.

Pearson correlation matrix with significance levels between soil physicochemical parameters and microbial functional populations
The relationship between environmental factors and the total populations of cellulolytic and lignocellulolytic microbes is illustrated in the Pearson correlation matrix shown in Figure 4, where correlation coefficients (r) are represented by color gradients.

The Pearson correlation results between environmental parameters and functional microbial populations show an informative ecological pattern, although not statistically significant due to sample limitations (n = 3). The total population of lignocellulolytic microbes (TP TA) exhibited a very strong negative correlation with N, P, and K, indicating that the lignin-decomposing group tends to decrease in soils with high nutrient availability. This pattern is consistent with findings in tropical forests that the addition of N or P can suppress ligninase enzyme activity and shift microbial composition, especially in phosphorus-poor forests.³² Nutrient manipulation studies in China have also reported that N input decreases the biomass of certain microbes and alters community balance, while P has a greater impact on P-limited ecosystems.³² On the other hand, cellulolytic microbes (TP CMC) show a moderate positive correlation with K, in line with the role of potassium-solubilizing microbes (KSM) that can enhance K availability and the activity of cellulose-degrading enzymes, including CMCase, as reported in various KSM inoculation studies.³³ The relationship between pH and temperature appears weaker, but remains physiologically relevant. Hydrolytic enzymes in tropical rainforest soils are known to have an optimum pH under acidic conditions, and edaphic changes can shift enzyme activity, as demonstrated in a soil enzyme study in Panama.^32,34 Long term warming has also been reported to alter the composition of microbial communities and carbon degradation.³⁵ Thus, although this correlation is not statistically significant, its ecological pattern remains consistent as an initial reference for the study.

Microbial isolation and enzyme activity based on colony diameter on specific media (CMC Agar and Tanat Agar)
Colony growth diameter observations indicated that isolate SP5 showed no growth on either selective medium (CMC Agar and Tanat Agar). This finding highlights the need for further investigation into several possible explanations: (1) cell dormancy, (2) specific nutrient requirements not provided by standard media, or (3) unique ecophysiological adaptations to the isolate’s native environment. This also underscores the complexity of microbial interactions with both abiotic and biotic factors in tropical rainforest ecosystems, which are currently mapped only spatially. Additional details are provided in Table 4.

Table 4. Colony Growth Characteristics and Lignocellulolytic and Cellulolytic Enzyme Activities of HPPB Soil Microbial Isolates on Selective Media

Description: Enzyme score: (-) absent, (+) low, (++) medium, (+++) high. SD: Standard deviation of three replicates. Incubation conditions: 36 °C, 72 hours, aerobic

The results provided information on the diversity of lignocellulose degradation ability in HPPB soil microbial isolates that showed different substrate specialization and biotechnological potential. Bacterial isolates SP2 and SP3 showed optimal growth on CMC Agar media with colony diameters of up to 84.1 ± 1.5 mm and 69.7 ± 1.2 mm respectively with high cellulase activity ( + + + and + + ). This finding is in line with the research of Ren et al.¹⁸ who reported that cellulolytic bacteria in tropical ecosystems tend to develop large colony growth strategies as a form of competitive adaptation in obtaining nutrients. However, SP2 which is only active on CMC Agar and does not show growth on Tanat Agar, this supports the concept of “metabolic trade-off” where microbes optimize one metabolic pathway at the expense of other capabilities.¹⁷

Furthermore, on Tanat Agar medium, fungal isolates SP6 and SP7 demonstrated lignocellulose-degrading capability, with colony diameters exceeding 80 mm and exhibiting the highest lignocellulase activity (+++). This pattern is consistent with the characteristics of white-rot fungi, particularly in relation to their complex ligninolytic enzyme system.³⁵ The dual growth capability of SP6 and SP7 on both media supports the hypothesis that tropical soil fungi have generally evolved lignocellulolytic capabilities as a survival strategy in highly competitive environments.¹⁷

From a biotechnological perspective, fungal isolates SP6 and SP7 exhibited strong lignocellulolytic activity on Tannic Acid (TA) agar, indicating their potential to degrade complex lignocellulosic substrates. Ligninolytic fungi are widely recognized for producing oxidative enzymes involved in lignin depolymerization and biomass conversion.³⁶ In contrast, isolate SP2 showed prominent cellulolytic activity on CMC agar, suggesting its ability to utilize cellulose as a carbon source through enzymatic degradation. Microbial cellulases play an important role in biomass transformation and industrial bioconversion processes.³⁷

The differences in functional activities among isolates reflect microbial adaptation to environmental conditions in tropical rainforest ecosystems. Such functional traits contribute to organic matter decomposition and carbon cycling processes.³⁸ The occurrence of lignocellulolytic fungi (SP6 and SP7) suggests a role in the degradation of plant-derived materials, whereas SP2 may contribute to cellulose decomposition and nutrient turnover in soil environments.³⁹

Nevertheless, the potential application of these isolates in enzyme production or lignocellulosic waste processing requires further validation under controlled and industry-relevant conditions.⁴⁰ In addition, isolate SP5 did not exhibit growth on either selective medium, which may indicate dormancy, specific nutritional requirements, or unsuitable cultivation conditions. Therefore, further physiological and molecular studies are necessary to clarify its ecological role and biotechnological potential. Overall, these findings highlight the diversity of microbial functional strategies in tropical rainforest ecosystems, as shown in Figure 5.

Visually, the bacterial colonies SP1, SP2, SP3, and SP4 exhibited similar coloration, ranging from white to cream, whereas the fungal isolates displayed more diverse pigmentation, with SP6 appearing green, SP7 white, and SP8 ranging from yellow to orange (Figure 5).

Molecular identification of bacterial and fungal isolates
The results of the molecular identification of bacterial and fungal isolates based on BLASTn analysis of the consensus sequences of the 16S rRNA gene (bacteria) and ITS (fungi) are presented in Table 5. Out of the eight isolates analyzed, only five produced consensus sequences of adequate quality for molecular identification. The displayed information includes sequence length, closest species match, percentage identity, query coverage, and final taxonomic decision. This data serves as a strong basis for validation before linking microbial identity with its functional potential, particularly related to lignocellulose degradation capability, soil microbial community dynamics, and their roles in decomposition processes and nutrient cycling in tropical environments.

Table 5. Molecular Identification Summary of Bacterial and Fungal Isolates

Note: Molecular identification of selected isolates based on BLASTn analysis, including sequence length, closest match, identity percentage, query coverage, and taxonomic assignment. Bacterial isolates were identified as Bacillus spp., while fungal isolates were classified as Trichoderma sp. and Penicillium/Aspergillus group.

Molecular identification shows that all bacterial isolates (SP1, SP2, and SP3) belong to the genus Bacillus, which is a group of bacteria that dominate tropical soils and play an active role in the decomposition of organic matter, nutrient cycling, and enzymatic degradation of lignocellulosic substrates.^18,37 High sequence identity values (≥98%) and strong query coverage in isolates SP1, SP2, and SP3 support the classification at the species level. In line with several references that Bacillus spp. often dominate nutrient-rich microhabitats, have high metabolic flexibility, and are capable of adapting to environmental fluctuations typical of tropical forest ecosystems.^10,38 Moreover, the presence of Bacillus among isolates with high enzymatic activity is consistent with many studies that show the genus has a strong ability to produce cellulase, xylanase, and lignin-degrading enzymes, which are very important in the biomass decomposition process.^30,37

For the fungal isolates JSP8 and JSP6, BLASTn analysis revealed close affiliations with the genera Aspergillus, Trichoderma, and Penicillium, which are recognized as important fungal groups involved in lignocellulose decomposition in tropical ecosystems. JSP8 was strongly identified as Trichoderma spp., consistent with the role of this genus as a major producer of cellulase, hemicellulase, and other lignocellulose-degrading enzymes, as well as its ability to compete in soil environments rich in plant polymers.⁷ Meanwhile, JSP6 was closely associated with Penicillium, which contributes to litter decomposition, nutrient mineralization, and the enzymatic breakdown of complex lignocellulosic polymers in tropical forest soils.¹⁹ Overall, these molecular findings confirm that the obtained isolates correspond to key microbial taxa involved in lignocellulose degradation and biochemical processes in tropical rainforest soils. These findings are further supported by their roles in fungal-mediated lignin biodegradation³⁹ and oxidative enzyme systems, including lignin peroxidase, manganese peroxidase, and laccase.⁴¹

Dynamics of cellulase and ligninolytic enzyme activities during Litter-Weed-Biochar decomposition and their biotechnological implications
The activity of cellulase and lignocellulase (LiP) enzymes from the eight purified isolates showed that only five isolates had optimal capabilities and were able to grow during purification. After the decomposition process of the litter-weeds-biochar for 28 days, the ability of the microbial isolates to degrade cellulose and lignocellulose components and their applicative potential in biomass-based biotechnology can be observed. Enzyme activity in each isolate can be seen in Tables 6 and 7.

Table 6. Comparative Temporal Profiling of Cellulase Activity Microbial Isolates from Tropical Rainforest Soils

Note: Cellulase enzyme activity of microbial isolates was measured over 28 days of litter–weed–biochar decomposition. Bacterial isolates SP1–SP3 were identified as Bacillus sp. (SP1 and SP2) and Bacillus group (SP3), while fungal isolates SP6 and SP8 belonged to the Penicillium/Aspergillus group (SP6) and Trichoderma sp. (SP8).
Different superscript letters within the same row indicate significant differences (p < 0.05), as determined by ANOVA followed by Duncan’s Multiple Range Test (DMRT)

Table 7. Comparative Temporal Profiling of Lignocellulolytic Activity Microbial Isolates from Tropical Rainforest Soils

Note: Lignocellulolytic enzyme activity of microbial isolates was measured over 28 days of litter–weed–biochar decomposition. Bacterial isolates SP1–SP3 were identified as Bacillus sp. (SP1 and SP2) and Bacillus group (SP3), while fungal isolates SP6 and SP8 belonged to the Penicillium/Aspergillus group (SP6) and Trichoderma sp. (SP8).

Different superscript letters within the same row indicate significant differences (p < 0.05), as determined by ANOVA followed by Duncan’s Multiple Range Test (DMRT)

Statistical analysis explains that the significance of the data shows that cellulase activity in bacterial isolates (SP1, SP2, SP3) is faster on the 7^th day, while fungal isolates (SP6, SP8) are optimal on the 14^th day. This pattern is consistent with the literature, which states that cellulase production by soil microorganisms generally reaches its peak within 5-7 days of incubation, coinciding with the availability of easily accessible cellulose substrate in the early fermentation phase. Relevant to controlled fermentation studies that report that the peak of enzymatic activity can occur in the first week of incubation, especially in soil cellulolytic microfungi and bacteria.⁴⁰ Bacteria are more effective in the initial decomposition of simpler fractions such as cellulose and hemicellulose, while fungi, especially the white rot fungi group, are more dominant in lignin oxidation with enzymes such as LiP, MnP, and laccase. Bacteria are more effective in the initial decomposition of simpler fractions such as cellulose and hemicellulose, while fungi, especially the white rot fungi group, are more dominant in lignin oxidation with enzymes such as LiP, MnP, and laccase. Research in the tropical forest soil of Puerto Rico also shows that bacteria can grow on substrates containing lignin and enhance the activity of enzymes such as phenol oxidase and peroxidase, although at the same time, cellulase activity decreases. Additionally, research in the Amazon and Atlantic forest regions found that tropical environments are an important source of lignocellulolytic bacteria and actinomycetes with great potential for biotechnological applications.⁴¹ The focus of biotechnology sees the combination of bacterial and fungal isolates synergizing to become more efficient in biomass bioconversion. Studies on lignocellulose degradation emphasize the importance of microbial consortia, where bacteria provide the initial stage of carbohydrate depolymerization, followed by fungi with the more complex oxidation of lignin. In line with reports on the gradual increase in degradation efficiency, and relevant for industrial applications such as bioethanol production, lignocellulosic biorefineries, and biomass waste processing.⁴²