Effects of Brown Sugar Supplementation on Fermentation Quality and Microbiota of Mixed Silage of Wheat Straw and Pennisetum giganteum

Technical Summary: Effects of Brown Sugar Supplementation on Fermentation Quality and Microbiota of Mixed Silage of Wheat Straw and Pennisetum giganteum

Problem Statement
Pennisetum giganteum is a high-yield perennial grass with significant potential for ruminant feed; however, its ensiling is often compromised by high buffering capacity and low water-soluble carbohydrate (WSC) content, leading to inadequate fermentation, slow pH decline, and poor silage quality. While mixing it with wheat straw offers physical and nutritional complementarity, both materials remain low in fermentable sugars. Although molasses has been studied as a sugar additive, direct research on brown sugar—a widely available, standardized, and easily stored alternative—remains limited. This study addresses the gap in understanding how varying levels of brown sugar supplementation affect the fermentation quality and microbial community dynamics of mixed Pennisetum giganteum and wheat straw silage.

Methodology
The study employed a controlled experimental design using a mixture of fresh Pennisetum giganteum and wheat straw (7:3 fresh-to-dry ratio). Five treatment groups were established by adding brown sugar at levels of 0 (T0), 2 (T2), 4 (T4), 6 (T6), and 8 g·kg⁻¹ (T8). Each treatment included three replicates. A self-developed microbial inoculum containing Lactobacillus plantarum, Lactobacillus buchneri, and cellulase was applied to all samples. The mixtures were vacuum-packed in polyethylene bags and fermented at room temperature (20–26°C) for 60 days.

Fermentation quality was assessed by measuring pH, moisture content, ammonia nitrogen (NH3-N), and organic acids (lactic, acetic, propionic, and butyric acids) via High-Performance Liquid Chromatography (HPLC). Microbial community structure was analyzed using high-throughput sequencing of the 16S rRNA gene (V3-V4 region) on the Illumina NovaSeq 6000 platform. Statistical analyses included ANOVA, PCoA, NMDS, and network topology analysis (using Gephi) to evaluate bacterial co-occurrence patterns based on Pearson correlations among the top 50 genera.

Key Results

1. Fermentation Quality: The addition of brown sugar significantly influenced organic acid profiles. The T6 group (6 g·kg⁻¹) exhibited the highest lactic acid (397.89 g/kg) and acetic acid (55.59 g/kg) contents, which were significantly higher than other groups (P < 0.01 and P < 0.05, respectively). Conversely, butyric acid levels in T6 and T8 were significantly lower than in the control (T0) and T2 groups, indicating reduced spoilage. Moisture content decreased progressively with increased sugar addition, while pH remained within a favorable range (3.8–4.2) across all treatments.
2. Microbial Diversity and Composition: Brown sugar addition did not significantly alter alpha diversity indices (Chao1, Shannon, Simpson) across treatments, though all groups showed high coverage (>0.99). However, beta diversity analyses (PCoA and NMDS) revealed a clear separation between the control (T0) and the T6 group, indicating a significant shift in overall community structure.
   • At the phylum level, the relative abundance of Fusobacteriota (associated with protein degradation) was significantly lower in all sugar-treated groups compared to T0.
   • At the genus level, Exiguobacterium was significantly more abundant in treated groups.
   • LEfSe analysis identified specific biomarkers: T0 was characterized by Fusobacteriota and related taxa, while T4 was characterized by Alcanivoracaceae and Alcanivorax.
3. Network Structure: Network analysis of bacterial co-occurrence showed that groups T4 and T6 possessed the highest topological complexity, with the maximum number of edges (166), average degree (11.067), and graph density (0.382). This suggests that appropriate sugar addition enhanced the cooperativity and tightness of interactions among bacterial genera, creating a more stable and efficient microbial network.

Key Contributions

• Optimization of Additive Dosage: The study identifies 6 g·kg⁻¹ as the optimal dosage of brown sugar for mixed Pennisetum giganteum and wheat straw silage, balancing substrate availability with microbial regulation.
• Microbial Mechanism Elucidation: The research provides evidence that brown sugar acts as a readily available carbon source that drives the proliferation of lactic acid bacteria, suppresses detrimental taxa like Fusobacteriota, and restructures the microbial co-occurrence network toward a tighter, more synergistic state.
• Differentiation from Molasses: By focusing specifically on brown sugar, the study offers data distinct from molasses-based research, addressing the specific compositional differences (e.g., mineral and nitrogen content) between the two additives.

Significance and Claims
The authors claim that the addition of an appropriate amount of brown sugar effectively improves the fermentation quality of mixed silage by promoting lactic acid accumulation and inhibiting butyric acid production. The study posits that the T6 treatment achieves an optimal balance where the microbial community evolves into a "tighter and more efficient fermentation network," characterized by high graph density and enhanced cooperative interactions among core genera.

The paper concludes that this approach provides a theoretical basis for optimizing ensiling techniques for Pennisetum giganteum and improving the utilization of agricultural by-products like wheat straw. The authors explicitly note limitations, including the use of a microbial inoculum in all treatments and the lack of assessment regarding aerobic stability or animal performance metrics. Consequently, the study frames its findings as a foundational step, suggesting that future research should validate the T6 treatment under aerobic exposure and in vivo feeding trials.