Bacteria from the termite gut as novel sources of lignocellulose-degrading enzymes

Abstract:

Termites have achieved an outstanding ecological success, with more than 3000 extant species widely distributed around the globe. Termites ingest lignocellulosic substrates at different degrees of decomposition, and they feed for example on wood, grass, and litter but also on highly-degraded wood. Termites are among the few animals capable of producing endogenous cellulolytic enzymes, but this capacity alone is insufficient to decompose plant biomass. The wood-feeding lower termites associate with cellulolytic flagellates and gut bacteria. Approximately 60 million years ago, the ancestor of the higher termites lost the gut flagellates and associated with an almost exclusively bacterial gut microbiota. Lignocellulose is the main diet of termites. This recalcitrant composite material is broken down in the termite gut by the help of a range of different bacteria in their gut, hence these organisms must be able to synthesis sufficient quantities and activities of enzymes involved in the depolymerisation of cellulose, hemicellulose as well as lignin, yet this aspect is scarcely studied to date. In this collaborative study between UGM and BOKU gut bacteria of wood-feeding termites (Rhinotermitidae and Termitidae) were isolated, and studied pertaining to their ability to form various lignocellulolytic enzymes (cellulases, laccases, and dye-decolorizing peroxidases).

Termites were collected from termite mounds at the arboretum of Unversitas Gadja Mada, Yogyakarta and identified by their characteristics marked in red:

(a) mandibular without teeth, © Putri Dwi Mulyani

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Implementation Period:

20.02.2023 – 04.03.2023

Project:

Termites have achieved an outstanding ecological success, with more than 3000 extant species in 281 genera and eight families that are widely distributed around the globe, including in tropical, subtropical and warm temperate regions. Termites are broadly divided into the lower (families: Mastotermitidae, Stolotermitidae, Hodotermitidae, Archotermopsidae, Kalotermitidae, Serritermitidae and Rhinotermitidae) and higher (family: Termitidae) termites based on the respective presence or absence of intestinal flagellates. Termites ingest lignocellulosic substrates at different degrees of decomposition, and they feed for example on wood, grass, and litter but also on highly-degraded wood and soil with a high organic content. Termites are among the few animals capable of producing endogenous cellulolytic enzymes, but this capacity alone is insufficient to decompose plant biomass. Thus, they rely on their symbiotic gut microflora to depolymerize lignocellulose with subsequent fermentation, resulting in the production of short-chain fatty acids that can be oxidized by the host. The wood-feeding lower termites associate with cellulolytic flagellates and gut bacteria, of which the most abundant are in the phyla Spirochaetes and Proteobacteria. Approximately 60 million years ago, the ancestor of the higher termites lost the gut flagellates and associated with an almost exclusively bacterial gut microbiota that contributes significantly to the deconstruction of lignocellulose ingested by these termites. Lignocellulose is the main diet of termites. This recalcitrant composite material is broken down in the termite gut by the help of a range of different bacteria as well as other microorganisms, hence these organisms must be able to synthesis sufficient quantities and activities of enzymes involved in the depolymerisation of cellulose, hemicellulose as well as lignin, yet this aspect is scarcely studied to date (Bugg et al. 2011).

Interestingly, enzymes from termite-associated microorganisms have been comparatively little studied, and thus form an untapped source of potentially attractive novel enzymes (Gragg at el. 2015). It was the aim of this project and the ongoing collaboration to study bacteria isolated from the gut of termites indigenous to Indonesia, to screen these isolates for their ability to form a range of different enzyme activities (cellulase, hemicellulases, ligninases, proteases, as well as various other oxidoreductases that are implicated in lignocellulose degradation, and to biochemically characterise these enzymes to some extent. In addition to different heme-containing peroxidases that are expected to be involved in lignin degradation we also suggest that a range of auxiliary enzymes that provide hydrogen peroxide to these peroxidases are formed by these bacteria. Our group at BOKU has studied these auxiliary enzymes, which are summarised as family AA3 enzymes in the CAZy database (http://www.cazy.org/Auxiliary-Activities.html), extensively over the last 15 years (Sützl et al. 2018), so we are particularly interested in these enzymes as well.

In this collaborative study between UGM and BOKU gut bacteria of wood-feeding termites (Rhinotermitidae and Termitidae) were isolated, and studied pertaining to their ability to form various lignocellulolytic enzymes (cellulases, laccases, and dye-decolorizing peroxidases (DyP)). Bacteria were first screened by using plate-based assays, and potential producers of these enzymes were identified based on 16S ribosomal RNA (16S rRNA) sequences as Bacillus thuringiensis, Lysinibacillus sphaericus, Achromobacter sp., Trabulsiella odontotermitis, Trabulsiella guamensis, Agrobacterium sp., and Citrobacter sp. When cultivated on a cellulose-based medium, Achromobacter sp. gave the highest cellulase activity, while growth on a lignin-based medium identified Lysinibacillus sphaericus as the best laccase producer. Furthermore, B. thuringiensis and Agrobacterium sp. were identified as potential producers of dye-decolorizing peroxidases (DyP), respectively.

Based on our results we concluded that the gut of the working termites from colonies of Rhinotermitidae and Termitidae contains bacteria capable of producing cellulase, laccase and dye-decolorizing peroxidase, which play a role in deconstructing lignocellulose and have important industrial applications. The activity of lignocellulolytic enzymes generally varies with the type of bacterial isolate, pH and temperature. Our joint research may open up the possibility to apply these industrially relevant enzymes and to study them in more detail.

In the course of the project stay at the UGM, the agreement on a cotutelle doctoral program of Mr. Arief Muammar, MSc, between the UGM and BOKU was prepared (it has by now been signed by both universities), and details of the scientific work program of this new doctoral project between UGM and BOKU were discussed and agreed. During his stay at BOKU (two years), Mr. Muammar will perform metagenome sequencing of stool samples of endemic Indonesian Bawean deer (Axis kuhlii) and investigate the diversity of the microbiome in these samples. Furthermore, sequences will be selected from these metagenome data, which – according to their sequence similarities – code for lignocellulose-degrading enzymes. These sequences will be introduced into suitable heterologous expression systems (technology available at BOKU) and the enzymes produced in this way will be biochemically characterized. Overall, this will be an extension of the collaborative research on lignocellulose-deconstructing enzymes from termites.

A similar approach, based on the preliminary work already carried out, will also be followed for the study of the metagenome of the intestinal contents of termites. This project will also offer young scientists from UGM the opportunity to complete part of their doctoral studies within the framework of a corresponding cotutelle doctorate, or to complete the entire doctoral program at BOKU through suitable financial support (Austrian-Indonesian scholarship program, ASEA-Uninet Ernst Mach Grant). Furthermore, lectures on recombinant protein production were held as part of an international master’s program (in English), in which these possibilities of personnel exchange were pointed out.

References

Bugg, T.D.H., Ahmad, M., Hardiman, E.M., Singh, R. (2011) The emerging role for bacteria in lignin degradation and bio-product formation. Curr. Op. Biotechnol. 22: 394-400

Cragg, S.M., Beckham, G.T., Bruce, N.C., Bugg, T.D.H., Distel, D.L., Dupree, P., Green Etxabe, A., Goodell, B.S., Jellison, J., McGeehan, J.E., McQueen-Mason, S.J., Schnorr, K., Walton, P.H., Watts, J.E.M., Zimmer, M. (2015) Lignocellulose degradation mechanisms across the Tree of Life. Curr. Op. Chem. Biol. 29: 108-119

Sützl, L., Laurent, C. V.F.P., Abrera, A.T., Schütz, G., Ludwig, R., Haltrich, D. (2018) Multiplicity of enzymatic functions in the CAZy AA3 family. Appl. Microbiol. Biotechnol. 102: 2477–2492

We are currently preparing the joint publication “Characterization of lignocellulolytic enzymes from gut bacteria of wood-feeding termites (Isoptera: Rhinotermitidae and Termitidae)” for submission. Authors: Putri Dwi Mulyani, Dietmar Haltrich, Clemens Karl Peterbauer, Yekti Asih Purwestri.