Bangladesh, with a land area of 147,570 square kilometers, is a predominantly agro-based developing country where agriculture remains a cornerstone of the national economy. Approximately 45% of the labor force is engaged in agricultural activities, contributing around 17% to the gross domestic product (GDP) [1]. Despite increasing industrialization and urban growth, the agricultural sector continues to play a pivotal role in employment generation, poverty reduction, food security, and rural development [2]. Over 70% of rural households still depend, wholly or partially, on agriculture for their livelihoods, highlighting its socio-economic relevance [3]. However, this vital sector is facing multiple challenges, including decreasing arable land, water scarcity, and the adverse impacts of climate change. Rapid urban expansion and population growth are intensifying pressure on already limited natural resources, raising serious concerns about the sustainability of food production. Bangladesh ranked 84th out of 113 countries in the 2023 Global Food Security Index, emphasizing the country’s vulnerability in achieving long-term food and nutritional security [4]. Environmental degradation further complicates this situation. Bangladesh has only 11.2% of its total land area covered by forests, making it one of the least forested countries in South Asia [5]. The country lost approximately 840,000 hectares of forest between 1990 and 2010, with a deforestation rate of 42,000 hectares per year, particularly in the semi-evergreen hill forests of Chittagong [6]. Additionally, sea-level rise, encroachment, and overexploitation are threatening ecologically critical zones such as the Sundarbans, the world’s largest mangrove forest [7]. Deforestation reduces biodiversity and carbon sequestration capacity, while exacerbating climate risks. As deforestation accounts for nearly 17% of global CO₂ emissions, it underscores the urgency for sustainable land management practices [8]. Agroforestry, the deliberate integration of trees with crops and/or livestock, is increasingly promoted as a sustainable land-use approach. Agroforestry systems offer numerous benefits, including improved soil health, enhanced biodiversity, microclimate regulation, diversified income sources, and resilience to climate variability [9]. In Bangladesh, agroforestry is gaining popularity as a practical strategy to ensure environmental sustainability and improve rural livelihoods [10]. Commonly used tree species include Mangifera indica, Zizyphus jujuba, Litchi chinensis, and Artocarpus heterophyllus, which also contribute to economic stability [11]. Leaf litter from these trees is a key source of organic matter and nutrient enrichment through microbial decomposition [12]. Fungi and bacteria break down the lignocellulosic biomass in leaf litter, which is mostly made up of cellulose, hemicellulose, and lignin. This allows nutrients to be released into the soil [13]. Bacterial genera such as Bacillus, Pseudomonas, Clostridium, Xanthomonas, and Flavobacterium are known for producing extracellular enzymes vital for this degradation process [11]. However, due to the complexity of plant material, microbial consortia—interacting communities of microbes—are more effective, offering higher efficiency, adaptability, and consistency under diverse conditions [14]. This study aims to explore the microbial diversity and degradative efficiency of leaf-litter-decomposing bacterial consortia in agroforestry systems of Bangladesh

Sample Collection and Materials

Mature leaves of guava (Psidium guajava), jackfruit (Artocarpus heterophyllus), lemon (Citrus limon), litchi (Litchi chinensis), and mango (Mangifera indica) were collected from trees located in Nirala, Rupsha, and Khulna University areas of Khulna, Bangladesh. The collected leaves were cleaned thoroughly with tap and distilled water, then air-dried before use. Essential equipment included an incubator, pH meter (Hanna Instruments, USA), digital balance (Ohaus, USA), micropipette (Gilson, France), atomic absorption spectrophotometer (SHIMADZU AA-7000, Japan), shaking incubator, centrifuge, spectrophotometer, and refrigerator. Standard glassware and chemicals such as peptone, beef extract, and sodium hydroxide (NaOH) were used throughout the study.

Isolation and Characterization of Bacteria

A stored bacterial consortium was thawed and inoculated into autoclaved nutrient broth, then incubated at 30°C for 24 hours. For isolation of pure colonies, the broth was streaked on nutrient agar plates and incubated under the same conditions. Distinct colonies were repeatedly streaked to ensure purity, and subcultured onto agar slants for preservation at –20°C. Bacterial isolates were characterized based on colony morphology (color, shape, opacity, elevation, and margin). Gram staining was performed following standard procedures to differentiate Gram-positive and Gram-negative bacteria. Motility was assessed using TTC-containing semi-solid agar; motile organisms displayed diffuse growth from the stab line, while non-motile strains remained confined.

Leaf Degradation Assay

Leaf samples were cut into ~1.5-inch pieces. For degradation assessment, 5 g of a mixed sample (1g from each species) was placed into sterile flasks. Each flask received 100µL of bacterial culture and 5 mL sterile distilled water and was incubated at 30°C for 21 days. Sterile water (5mL) was added every five days. Post-incubation, 45mL sterile water was added, and the contents were filtered using Whatman No. 1 paper. The retained biomass was dried at 105°C to determine the remaining mass.

Moisture and Dry Matter Analysis

Fresh weights of leaves were recorded after washing and drying. Moisture content was calculated by drying samples in an oven at 105°C to constant weight using the formula:

Moisture (%) = (Initial weight - Final weight - Initial weight) \ Initial weight ×100 

Dry matter (%) was determined by subtracting the moisture percentage from 100.

Determination of Pure Culture of Bacteria on Agar Plate

To isolate pure bacterial colonies, the streak plate method was employed. Distinct colony morphologies were observed on nutrient agar plates, indicating successful isolation of different bacterial strains from the mixed population. The colonies varied in shape, opacity, color, elevation, and margin, as summarized in Table 1. Most isolates exhibited a circular or round morphology with convex elevation and entire margins, while a few showed curled margins or flat elevation. Pigmentation ranged from white, off-white, yellow, and red to soft yellow. Notably, isolates 6 and 9 produced clear colonies with soft yellow pigmentation.

Table 1: Cultural Characteristics of Isolated Colonies on Agar Plate

Subculturing on Agar Slants

To preserve and maintain the purity of isolates, subculturing was performed on agar slants. The isolates displayed distinct growth patterns, including filiform, beaded, and diffuse or spreading forms. Pigmentation observed on the slants (Table 2) was consistent with that on the agar plates, further confirming the stability of these traits.

Table 2: Cultural Characteristics of Isolated Colonies on Agar Slant

Morphological Characterization

Gram Staining Test

Microscopic observation following Gram staining revealed that eight of the nine bacterial isolates were Gram-negative, exhibiting pink coloration, while isolate 9 was Gram-positive, retaining the crystal violet stain. Most isolates (1, 3, 6, 7, 8) showed coccoid shapes occurring singly. Isolate 2 exhibited rod-shaped, short chains characteristic of Proteobacteria. Isolate 5 presented a mixed morphology—coccoid and occasional rods. Isolate 9 displayed coccoid cells in short chains, suggestive of Gram-positive Streptococcus species. No flagella were detected in any of the isolates, indicating a lack of motility (Table 3).

Table 3: Morphological Characteristics of Isolated Bacteria After Gram Staining

Motility Test

All bacterial isolates tested negative for motility, as evidenced by growth restricted to the stab line in motility agar. This further supports the absence of flagellar structures, aligning with the Gram staining results.

Moisture Content and Dry Matter Degradation

Mixed leaves from five plant species showed an average moisture content of 52% and dry matter content of 48%. Upon inoculation with isolated bacterial strains and incubation for 21 days, dry matter degradation was evaluated. Among all isolates, isolate 4 exhibited the highest degradation efficiency. Figure 1 illustrates the extent of dry matter loss, with significant variation among treatments (P < 0.05). The mixed bacterial consortium also showed notable degradation, though slightly less than isolate 4.

Figure 1: Mass degradation (g) of plant leaves with bacteria. Values are presented as mean ± SD. Different letters (a-f) indicate significant differences at P < 0.05. Here treatments denote individual bacterial isolates (1-9) & and 10 defines (mix of all isolates,1-9) .C denotes control; leaves degradation without bacteria.

Microbial decomposition of lignocellulosic leaf litter plays a vital role in nutrient cycling and carbon turnover in terrestrial ecosystems, particularly within agroforestry systems that integrate fruit trees into agricultural landscapes [15].  In this study, bacterial isolates obtained from soil environments demonstrated significant variation in their ability to degrade mixed fruit tree leaves (Mangifera indica, Artocarpus heterophyllus, Citrus limon, Litchi chinensis, and Psidium guajava). Among the nine isolates tested, isolate 4 showed the highest degradation efficiency, achieving a 47% reduction in dry matter, followed by isolate 6 with 36% degradation. The bacterial consortium exhibited a moderate but notable effect, suggesting possible antagonistic or synergistic interactions among strains [16]. These findings underscore the complexity of microbial consortia in biodegradation processes and highlight the potential of specific isolates for application in sustainable leaf litter management [17]. The observed differences in degradation efficiency may be attributed to variations in enzyme production, substrate specificity, and microbial metabolism. Previous studies have reported that individual bacterial strains often lack the metabolic breadth to effectively decompose complex lignocellulosic biomass, resulting in slower degradation rates [18]. In contrast, microbial consortia, particularly those derived from diverse soil environments, have demonstrated enhanced degradation performance due to metabolic cooperation and division of labor [19]. Our results partially support this, with isolate 4 outperforming the mixed consortium, possibly due to superior enzyme secretion or faster colonization. However, the consortium still performed better than many individual strains, validating its potential use where single-strain efficacy is insufficient or unpredictable under environmental variability [20]. The cultural characteristics of bacterial isolates on agar plates and slants revealed significant morphological diversity, with colonies varying in pigmentation, elevation, and margin. Isolates 6 and 9, which produced soft yellow colonies, were particularly notable for their pigment stability across media. Pigmentation in bacteria is often linked to oxidative stress tolerance and environmental adaptation, which could indirectly influence their survival and functional performance during the degradation process [21]. Interestingly, while isolate 6 showed strong pigment production and moderate degradation capacity, isolate 9 despite being the only Gram-positive strain had lower degradation efficacy, possibly due to limited extracellular enzyme expression or slower growth kinetics. The Gram staining results indicated that the majority of isolates were Gram-negative and coccoid in morphology, with isolate 2 being the only rod-shaped strain. No motility was observed in any isolates, which aligns with their lack of flagella. While motility is not a prerequisite for litter degradation, non-motile bacteria may rely more on direct substrate contact and biofilm formation to sustain their activity [22]. This could influence their colonization pattern on leaf surfaces and ultimately affect their degradation efficiency.

Compared with previous literature, the degradation percentages achieved in this study are consistent with earlier work on lignocellulose degradation by soil bacteria. Soil-derived Bacillus and Pseudomonas species could degrade cellulose by 30–50%, depending on the substrate and incubation period [23]. Similarly, Gu et al. (2013) isolated ligninolytic bacteria from wetland environments capable of degrading lignin-rich substrates by up to 40% within four weeks [24]. Our findings, particularly for isolate 4, are within this range and indicate that certain native soil bacteria have strong potential for biotechnological application in biomass decomposition. It is also important to highlight that the degradation performance in our study was measured after 21 days, aligning with standard timeframes used in similar investigations. The temperature during incubation although not specified here likely played a facilitating role, as microbial activity tends to increase with temperature up to an optimum range (typically 30–37°C for mesophilic bacteria). This agrees with observations from Ingraham & Bailey (1995) and Michie et al. (2011), who emphasized the temperature sensitivity of microbial decomposition processes [25,26]. The use of fruit tree leaves from agroforestry systems in this experiment is particularly significant. These trees Mangifera indica, Artocarpus heterophyllus, Citrus limon, Litchi chinensis, and Psidium guajava are widely cultivated in Bangladesh and other tropical countries, contributing both economically and ecologically. Their leaf litter is rich in lignocellulosic material, and without adequate microbial degradation, this biomass can accumulate, leading to soil surface hardening, pest proliferation, or nutrient immobilization [27]. Our study offers a practical solution by identifying native bacterial strains with the capacity to reduce this biomass load while returning nutrients to the soil. The ecological benefit is further amplified when considering that efficient litter decomposition reduces the need for chemical fertilizers and enhances soil organic carbon content [28]. Moreover, the enzymatic implications of these findings are noteworthy. Although enzyme assays were not conducted in this study, literature suggests that efficient leaf degradation is associated with enzymes such as cellulases, xylanases, and ligninases. Genera like Bacillus, Pseudomonas, and Flavobacterium are known producers of such enzymes [29]. The efficiency of isolate 4 may reflect the presence of one or more of these enzymatic activities, which could be explored in future studies through biochemical or genomic characterization. Additionally, the potential biotechnological applications of these isolates such as composting enhancers, biofertilizers, or components of integrated waste management systems warrant further exploration [30]. The control group (non-inoculated leaves) exhibited minimal degradation, confirming that microbial activity was the key driver of biomass loss. This reaffirms the importance of microbial inoculation in accelerating natural decomposition processes, especially in managed agroecosystems where organic load and nutrient turnover need to be optimized [31]. The variation in degradation across isolates further emphasizes the need for selective enrichment or genetic engineering to enhance microbial consortia tailored for specific leaf types or environmental conditions [32].

Limitations of the study: 

• Species-level identification of bacterial isolates was not performed.

• Enzymatic activity of isolates involved in degradation was not assessed.

• Microbial interactions within the consortium were not explored.

• The study was limited to short-term laboratory conditions.

• No field trials were conducted to validate degradation efficacy.

• Microbial growth kinetics during degradation were not monitored.

Soil-derived bacterial isolates showed promising potential for degrading mixed leaves from fruit trees, contributing to nutrient cycling and organic waste management in sustainable agriculture. Isolate 4 achieved the highest degradation rate, followed by isolate 6, indicating strong lignocellulose-degrading capability. The use of microbial consortia proved beneficial in accelerating biomass breakdown. These results highlight the practical relevance of leveraging native bacteria in agroforestry and composting practices. While molecular identification and enzymatic profiling remain necessary for deeper insights, the current findings provide a useful foundation for future research aimed at developing eco-friendly biotechnological applications in agroecological systems.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the Institutional Ethics Committee.

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