Screening of Phytophagous and Xylophagous Insects Guts Microbiota Abilities to Degrade Lignocellulose in Bioreactor

Abstract

Microbial consortia producing specific enzymatic cocktails are present in the gut of phytophagous and xylophagous insects; they are known to be the most efficient ecosystems to degrade lignocellulose. Here, the ability of these consortia to degrade ex vivo lignocellulosic biomass in anaerobic bioreactors was characterized in term of bioprocess performances, enzymatic activities and bacterial community structure. In a preliminary screening, guts of Ergates faber (beetle), Potosia cuprea (chafer), Gromphadorrhina portentosa (cockroach), Locusta migratoria (locust), and Gryllus bimaculatus (cricket) were inoculated in anaerobic batch reactors, in presence of grounded wheat straw at neutral pH. A short duration fermentation of less than 8 days was observed and was related to a drop of pH from 7 to below 4.5, leading to an interruption of gas and metabolites production. Consistently, a maximum of 180 mg_eq.COD of metabolites accumulated in the medium, which was related to a low degradation of the lignocellulosic biomass, with a maximum of 5 and 2.2% observed for chafer and locust gut consortia. The initial cell-bound and extracellular enzyme activities, i.e., xylanase and β-endoglucanase, were similar to values observed in the literature. Wheat straw fermentation in bioreactors leads to an increase of cell-bounded enzyme activities, with an increase of 145% for cockroach xylanase activity. Bacterial community structures were insect dependent and mainly composed of Clostridia, Bacteroidia and Gammaproteobacteria. Improvement of lignocellulose biodegradation was operated in successive batch mode at pH 8 using the most interesting consortia, i.e., locust, cockroaches and chafer gut consortia. In these conditions, lignocellulose degradation increased significantly: 8.4, 10.5, and 21.0% of the initial COD were degraded for chafer, cockroaches and locusts, respectively in 15 days. Consistently, xylanase activity tripled for the three consortia, attesting the improvement of the process. Bacteroidia was the major bacterial class represented in the bacterial community for all consortia, followed by Clostridia and Gammaproteobacteria classes. This work demonstrates the possibility to maintain apart of insect gut biological activity ex vivo and shows that lignocellulose biodegradation can be improved by using a biomimetic approach. These results bring new insights for the optimization of lignocellulose degradation in bioreactors.

Keywords: biomimetism; fermentation; insect guts; microbial diversity; volatile fatty acids.

FIGURE 1

Production and repartition of volatile fatty acids (VFA) and gasses in mg_eq.COD observed in (A) blank reactors -BR- and (B) wheat straw reactors –WSR. Measured VFA are acetate (C2), propionate (C3), butyrate and its isomer (C4 and iC4), and valerate and its isomer (C5 and iC5). Measured gasses are hydrogen (H₂) and methane (CH₄). WSR reactors were fed with wheat straw and BR contained insect gut without wheat straw.

FIGURE 2

Xylanase (A) and CMCase (B) activities in U.L-1 before the fermentation (1) and after the process for WSR (2), and for BR (3). Dark gray represents cell-bound activities (pellet) and light gray the free enzymatic activities (supernatant). WSR reactors were fed with wheat straw and BR contained insect gut without wheat straw.

FIGURE 3

Relative abundance of bacteria phylum (A) and bacteria class (B) identified from the Illumina Miseq sequencing data, within insect gut microbiomes before (T0) and at the end of cultures in batch anaerobic reactors: duplicate WSR1 and WSR2 reactors were fed with wheat straw and BR contained insect gut without wheat straw.

FIGURE 4

Diversity among bacterial community present within insect gut microbiomes before (T0) and after (WSR and BR) cultures in batch mode anaerobic reactors. Shannon diversity index evaluated at 97% similarity.

FIGURE 5

Principal coordinate analysis (PCoA) plot showing similarity relationships among bacterial community sampled from three groups of insect bioreactors (36% of the total variance explained).

FIGURE 6

Production and repartition of volatile fatty acids (VFA) in successive batch reactors inoculated with (A) cockroach (B) locust and (C) chafer microbiome in mg_eq.COD. Measured VFA are acetate (C2), propionate (C3), butyrate and its isomer (C4 and iC4), and valerate and its isomer (C5 and iC5).

FIGURE 7

Total xylanase and CMCase activity measured for WS-SBR-1 at day 0 (dark gray) and day 7 (white).

FIGURE 8

Relative abundance of bacteria phylum (A) and bacteria class (B) identified from the Illumina Miseq sequencing data, within insect gut microbiomes before (T0) and after cultures in successive batch reactors maintained at pH8: duplicate WS-SBR-1 and WS-SR-2 were fed with wheat straw and BR-SBR contained insect gut without wheat straw among two successive batch.

FIGURE 9

Principal coordinate analysis (PCoA) plot showing similarity relationships bacterial communities present within insect gut microbiomes before (T0) and at the end (WS-SBR and B-SBR) of cultures in successive batch reactors maintained at pH8. PCoA was conducted given the Hellinger distance matrix.

FIGURE 10

Diversity among bacterial communities present within insect gut microbiomes before (T0) and at the end of (WS-SBR-1, WS-SBR-2, and BR-SBR) cultures in successive batch reactors maintained at pH8. Shannon diversity index evaluated at 97% similarity.