This study was conducted to evaluate the potential of hydrolysable tannin (chestnut tannin, CHT) without or with condensed tannin (quebracho tannin, QT) for modulating alfalfa silage fermentation characteristics and in vitro ruminal methane (CH₄) production, fermentation profile, and microbiota. Alfalfa (235 g/kg fresh weight) was ensiled with no tannins (control), 2% CHT (CHT2), 5% CHT (CHT5), the combination of CHT and QT at 1% each (CHQ2), and CHT and QT at 2.5% each (CHQ5) of forage dry matter (DM). The CHQ2 treatment was more effective in reducing DM losses, pH, and ammonia–nitrogen to total nitrogen ratios of alfalfa silage than CHT2 and CHT5 treatments. All tannin treatments decreased ruminal CH₄ production, and the magnitude of the decrease was greater for the combinations than the individual ones. Total volatile fatty acid (VFA) concentrations and DM degradation decreased by tannin treatments, but microbial protein (MCP) synthesis increased. The total VFA concentrations and DM degradation were lower with CHQ2 treatment than with CHT5 and CHQ5 treatments, but the MCP concentrations were comparable among these treatments. Tannin inclusion decreased the abundance of the anaerobic fungi Ruminococcus albus and Ruminococcus flavefaciens, but enhanced Fibrobacter succinogenes. The combination of CHT and QT alleviated the inhibition of CHT supply alone in Butyrivibrio fibrisolvens, Ruminobacer amylophilus, and Prevotella ruminicola as well as protease. The results revealed that a combination of HT from CHT and CT from QT at a low level can reduce proteolysis and CH₄ production of alfalfa silage without impairing ruminal fermentation and microbiota.

Indole and skatole (3-methylindole, C₉H₉N) are common nitrogen-containing heterocyclic pollutants found in waste, wastewater treatment plants, and public restrooms and are the most notorious compounds in animal feces. Biodegradation was considered a feasible method for the removal of indole and skatole, but a comprehensive understanding of the metabolic pathways under both aerobic and anaerobic conditions was lacking, and the functional genes responsible for skatole biodegradation remained a mystery. Through metagenomic and gene cluster functional analysis, Acinetobacter piscicola p38 (NCBI: CP167896), genes 1650 (styrene monooxygenase: ACDW34_08180), and 1687 (styrene monooxygenase: ACDW34_08350) were identified as having the potential to degrade indole and skatole. The heterologous expression results demonstrate that the genes 1650 and 1651 (flavin reductase: ACDW34_08185), when combined, are capable of degrading indole, while the genes 1687 and 1688 (flavin reductase: ACDW34_08355), in combination, can degrade indole as well as skatole. These reactions necessitate the involvement of flavin reductase and NAD(P)H to catalyze the oxygenation process. This work aimed to provide new experimental evidence for the biodegradation of indole and skatole. This study offered new insights into our understanding of skatole degradation. The Acinetobacter_piscicola p38 strain provided an effective bacterial resource for the bioremediation of fecal indole and skatole.

Skatole is a harmful, odorous pollutant in livestock manure. Skatole-degrading strains are mostly harmful Gram-negative bacteria, whereas safe Gram-positive strains demonstrate poor degradation performance, limiting bioremediation applications. To solve this problem, this study was conducted to enhance the skatole degradation capacity of the food-grade strain Lactococcus lactis NZ9000 via heterologous expression of the skatole-degrading Ska enzyme from Acinetobacter piscicola p38. Three gene sequences, designated Ska-Y (original sequence from A. piscicola p38), Ska-D (E. coli codon-optimized), and Ska-R (L. lactis codon-optimized), were separately expressed using constitutive pMG36e and nisin-inducible pNZ8148 plasmids. The constitutive system only transcribed mRNA but produced misfolded, nonfunctional inclusion bodies. By contrast, the codon-optimized pNZ8148-Ska-R strain exhibited prominent skatole degradation ability, even under non-inductive conditions. Under optimal conditions (30 ℃, ultra-low nisin induction), the engineered strain increased the 24 h degradation rate of 50 mg/L skatole from 20% to 88% and completely degraded 25 mg/L skatole. Field tests verified that the strain effectively reduced skatole accumulation in manure compost and lagoon manure. This study achieved efficient functional heterologous expression in Gram-positive bacteria, provides new insights into ultra-low-dose induction and codon optimization, and offers a safe and efficient microbial agent for livestock manure odor remediation.