Isolation of lignocellulosic biomass-degrading bacteria from Porcellio dilatatus gut-enriched cultures

Abstract

The lignocellulosic biomass (LCB) is an attractive, sustainable, and environmentally friendly alternative to fossil sources to produce biofuel, biomaterials, and biochemicals. However, its recalcitrant and heterogenous structure challenges its biodegradation and valorization. The gut microbiome of soil invertebrate species has emerged as a rich source of LCB-degrading bacteria and enzymes in terrestrial ecosystems. The primary objective of this investigation was to identify the bacterial communities within the Porcellio dilatatus gut (Crustacea: Isopods), to obtain enriched cultures, and to identify bacterial isolates with LCB-degrading activity. A total of 112 enriched cultures were screened, all exhibiting xylanolytic activity. Among them, 94 displayed cellulolytic activity, 30 showed chitinolytic activity, and 21 demonstrated ligninolytic activity. Four enriched cultures were selected, and 128 bacteria with cellulolytic, xylanolytic, chitinolytic, or ligninolytic activity were isolated and taxonomically classified. The obtained results reinforce the potential of bacterial communities within the digestive tract of soil invertebrates as a valuable source of lignocellulose-degrading microorganisms. Thirty-one isolates underwent in-depth enzymatic characterization, and five were selected and functionally evaluated. An artificial bacterial consortium was constructed to assess the potential benefits of using consortia to achieve enhanced LCB degradation. The positive results of this proof-of-concept artificial consortium (PdG-AC) can be used in future applications and is a valuable tool for enzymatic and microbial consortia engineering by, e.g., changing growth conditions for enhanced LCB-degrading abilities. KEY POINTS: • The gut microbiome of Porcellio dilatatus was characterized. • Porcellio dilatatus gut hosts many lignocellulose-degrading bacteria. • Developed an artificial bacterial consortium for lignocellulose degradation.

Keywords: Porcellio dilatatus; Artificial consortium; Culture-dependent methods; Enriched bacterial cultures; Gut microbiota; LCB-degrading activity-matrix-database.

Fig. 1

Heatmap of the index of enzymatic activity (IEA) for carboxymethylcellulose, xylan, chitin, and guaiacol of enriched cultures obtained from P. dilatatus guts microbiota from A National Forest of the Choupal and B Botanical Garden of the University of Coimbra. Seven generations (G2-G8) are represented after growing in four substrates (carboxymethylcellulose (CMC), xylan (Xyl), chitin (Chit), and Biomass waste (BW). The IEA was classified as low (halo width 0.10–1.24 cm), medium (halo width 1.25–2.50 cm), and high (halo width > 2.50 cm). The low IEA values are highlighted in yellow; the medium IEA are highlighted in orange, and the high IEA values are highlighted in red

Fig. 2

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of the 128 representative strains. Isolates recovered for their cellulolytic activity are identified in red, xylanolytic activity in blue, chitinolytic in green, and ligninolytic activity in brown (guaiacol) and orange (2,6-DMP)

Fig. 3

Enzymatic activity matrix of the 31 selected representative strains against five substrates (carboxymethylcellulose (CMC), xylan (Xyl), chitin (Chit), 2,6-Dimethoxyphenol (DMP) and guaiacol (Gua) at three pH values. The IEA (index of enzymatic activity) was classified as low (0.10–1.24 cm), medium (1.25–2.50 cm), and high (> 2.50 cm). The low IEA values are highlighted in yellow; the medium IEA are highlighted in orange, and the high IEA values are highlighted in red

Fig. 4

Index of enzymatic activity (IEA) values matrix of the five single strains versus PdG-AC consortium for five substrates, carboxymethylcellulose (CMC), xylan, chitin, guaiacol, and 2,6-DMP at three pH values. The IEA was classified as low (0.10–1.24 cm), medium (1.25–2.50 cm), and high (> 2.50 cm). The low IEA values are highlighted in yellow; the medium IEA are highlighted in orange, and the high IEA values are highlighted in red. nd, not determined