Abstract
Traditionally, starting inoculants have been applied to improve ensiling of forage used for livestock feed. Here, we aimed to build up a bioinoculant composed of lactic acid-producing and lignocellulolytic bacteria (LB) derived from the Megathyrsus maximus (guinea grass) phyllosphere. For this, the dilution-to-stimulation approach was used, including a sequential modification of the starting culture medium [Man, Rogosa, and Sharpe (MRS) broth] by addition of plant biomass (PB) and elimination of labile carbon sources. Along 10 growth-dilution steps (T1–T10), slight differences were observed in terms of bacterial diversity and composition. After the sixth subculture, the consortium started to degrade PB, decreasing its growth rate. The co-existence of Enterobacteriales (fast growers and highly abundance), Actinomycetales, Bacillales, and Lactobacillales species was observed at the end of the selection process. However, a significant structural change was noticed when the mixed consortium was cultivated in higher volume (500ml) for 8days, mainly increasing the proportion of Paenibacillaceae populations. Interestingly, Actinomycetales, Bacillales, and Lactobacillales respond positively to a pH decrease (4–5), suggesting a relevant role within a further silage process. Moreover, gene-centric metagenomic analysis showed an increase of (hemi)cellulose-degrading enzymes (HDEs) during the enrichment strategy. Reconstruction of metagenome-assembled genomes (MAGs) revealed that Paenibacillus, Cellulosimicrobium, and Sphingomonas appear as key (hemi)cellulolytic members (harboring endo-glucanases/xylanases, arabinofuranosidases, and esterases), whereas Enterococcus and Cellulosimicrobium have the potential to degrade oligosaccharides, metabolize xylose and might produce lactic acid through the phosphoketolase (PK) pathway. Based on this evidence, we conclude that our innovative top-down strategy enriched a unique bacterial consortium that could be useful in biotechnological applications, including the development/design of a synthetic bioinoculant to improve silage processes.
Highlights
Ensiling is an ancient technique used to preserve nutrients in roughages, generally offered as a ruminant livestock feed (Ávila and Carvalho, 2019)
M. maximus like many tropical grasses contains a large amount of fiber, including lignin and structural carbohydrates linked to ferulic acid, preventing its efficient utilization directly in feedlots (Grabber et al, 2009)
It has been suggested that the inoculation of bacterial isolates and/or mixed microbial consortia (e.g., lactic acid bacteria (LAB) plus lignocellulolytic species) with the potential to produce cellulases, arabinofuranosidases, xylosidases, ferulic acid esterases (FAE; EC 3.1.1.73), and cutinases, could enhance the quality of the silage by increasing the availability of carbohydrates with lower complexity (Muck et al, 2018; Tarraran and Mazzoli, 2018; Bonaldi et al, 2021)
Summary
Ensiling is an ancient technique used to preserve nutrients in roughages (e.g., grass and legumes), generally offered as a ruminant livestock feed (Ávila and Carvalho, 2019) This process is based on spontaneous fermentation, where epiphytic lactic acid bacteria (LAB) metabolize plant-derived sugars, quickly decreasing the pH (between 5 and 4) and preventing silage spoilage (Fabiszewska et al, 2019). It has been suggested that the inoculation of bacterial isolates and/or mixed microbial consortia (e.g., LAB plus lignocellulolytic species) with the potential to produce cellulases, arabinofuranosidases, xylosidases, ferulic acid esterases (FAE; EC 3.1.1.73), and cutinases, could enhance the quality of the silage by increasing the availability of carbohydrates with lower complexity (Muck et al, 2018; Tarraran and Mazzoli, 2018; Bonaldi et al, 2021). FAE has been recently reported in LAB, and its use may show a potential improvement on neutral detergent fiber (Xie et al, 2021)
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