Different Inocula Produce Distinctive Microbial Consortia with Similar Lignocellulose Degradation Capacity
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Appl Microbiol Biotechnol DOI 10.1007/s00253-016-7516-6 ENVIRONMENTAL BIOTECHNOLOGY Different inocula produce distinctive microbial consortia with similar lignocellulose degradation capacity Larisa Cortes-Tolalpa1 & Diego Javier Jiménez1 & Maria Julia de Lima Brossi1 & Joana Falcão Salles1 & Jan Dirk van Elsas1 Received: 1 February 2016 /Revised: 21 March 2016 /Accepted: 24 March 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Despite multiple research efforts, the current strat- prevailed, in soil-derived ones β-glucosidases, and in egies for exploitation of lignocellulosic plant matter are still sediment-derived ones several activities. Isolates recovered far from optimal, being hampered mostly by the difficulty of from the consortia showed considerable metabolic diversities degrading the recalcitrant parts. An interesting approach is to across the consortia. This confirmed that, although the overall use lignocellulose-degrading microbial communities by using lignocellulose degradation was similar, each consortium had a different environmental sources of microbial inocula. unique enzyme activity pattern. Clearly, inoculum source was However, it remains unclear whether the inoculum source the key determinant of the composition of the final microbial matters for the degradation process. Here, we addressed this degrader consortia, yet with varying enzyme activities. Hence, question by verifying the lignocellulose degradation potential in accord with Beyerinck’s, Beverything is everywhere, the of wheat (Triticum aestivum) straw by microbial consortia environment selects^ the source determines consortium generated from three different microbial inoculum sources, composition. i.e., forest soil, canal sediment and decaying wood. We select- ed these consortia through ten sequential-batch enrichments Keywords Lignocellulose . Degrader communities . by dilution–to–stimulation using wheat straw as the sole car- Bacterial–fungal consortia . (Hemi) cellulolytic activity . bon source. We monitored the changes in microbial composi- Recalcitrant substrate tion and abundance, as well as their associated degradation capacity and enzymatic activities. Overall, the microbial con- sortia developed well on the substrate, with progressively- Introduction decreasing net average generation times. Each final consor- tium encompassed bacterial/fungal communities that were Lignocellulosic substrates such as wheat, grass and maize distinct in composition but functionally similar, as they all straws constitute important carbon and energy sources for mi- revealed high substrate degradation activities. However, we croorganisms. In addition to diverse small molecules, cellu- did find significant differences in the metabolic diversities lose and hemicellulose, both of which can be hydrolyzed to per consortium: in wood-derived consortia cellobiohydrolases sugars for further biological or chemical utilization (Xu et al. 2013), and lignin constitute the major carbonaceous compo- nents in these substrates. Whereas high-value products such as Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-7516-6) contains supplementary material, biofuels and diverse building blocks for industrial products which is available to authorized users. can be produced on the basis of the released compounds, the lignin moiety—a polymer of aromatic compounds like phe- * Larisa Cortes-Tolalpa nol—constitutes an important source material for the industri- [email protected] al production of adhesive resin and lignin gels. In plant tissue, the three moieties form a complex structure with intricate 1 Genomics Research in Ecology and Evolution in Nature, Groningen bonds, part of which is very difficult to breakdown. Thus, Institute for Evolutionary Life Sciences, University of Groningen, despite multiple research efforts, the current strategies for ex- Nijenborgh 7, 9747, AG Groningen, The Netherlands ploitation of lignocellulosic plant matter are still far from Appl Microbiol Biotechnol optimal, being hampered mostly by the challenge of the 100-year old Beijerinck adagium BEverything is every- degrading the recalcitrant parts of all three moieties. where and the environment selects^. In natural systems, lignocellulose degradation is carried out Here, we investigated whether different source communi- by multiple—coexisting—lignocellulolytic microorganisms. ties would generate similar lignocellulolytic microbial consor- These include a range of fungi and bacteria capable of pro- tia when similar selection criteria are applied. Whether this ducing a variety of degrading enzymes. These microorgan- adagium would be turned into reality presumably depends isms most likely establish synergistic relationships among on (1) the nature of the source inocula and (2) the strength each other and/or with other, non-cellulolytic, microbial of the selective force applied. Thus, the aims of this study were species and these interactions are expected to enhance the (i) to determine the relevance of the microbial inoculum as the substrate degradation rates (Lynd et al. 2002). For instance, driver of the lignocellulose-degradative consortia produced in forest soils, fungal and bacterial communities play impor- after ten enrichment steps and (considering the high degree tant roles; the former explore dead plant matter even at low of functional redundancy often observed in microbial commu- moisture content of the soil and the latter may act as secondary nities) (ii) to assess whether different source inocula result in responders (Lynd et al. 2002). Similarly, in decaying wood, similar degradation capacities. To this end, three different fungi act together with bacteria, constituting the communities source inocula, i.e. microbiomes from forest soil, canal sedi- that collectively work on the substrate (Prewitt et al. 2014) ment and decaying wood, were used for serial-batch dilution- whereas in sediment, we expect anaerobic cellulolytic to-stimulation on severed wheat straw as the carbon- and bacteria, possibly concomitant with particular fungi, to be energy-yielding substrate, in order to measure their effects involved in the biopolymer degradation processes (Wei et al. on the final enriched consortia. 2009). Although cooperation between microbes seems to be the driving force behind lignocellulose degradation in natural habitats, in industry single strains are often used (Guerriero Materials and methods et al. 2015). Using microbial consortia instead of single strains for Substrate preparation the biodegradation of lignocellulose allows one to take advantage of the microbial interactions, by making opti- Wheat straw used as the substrate was obtained from a local mal use of their intricate regulatory systems, which may farm (Groningen, the Netherlands). It was air-dried (50 °C) bypass problems of feedback regulation and metabolite before cutting it into pieces of about 5 cm length. Then, the repression that are often posed by single strains pieces were mixed with sterile distilled water and thoroughly (Wongwilaiwalin et al. 2010). Additionally, this strategy ground, using a mill hammer, to pieces ≤1mminorderto may confer an optimal combination of enzyme production increase the surface to volume ratio. After this treatment, the and interaction of microbes. Enrichment culturing—also wheat straw suspension was autoclaved at 121 °C for 27 min coined Bhabitat biasing^ (Ekkers et al. 2012)—is a strate- before use. Sterility of the substrate was verified following gy in which a deliberate Bbias^ is introduced into an en- plating on LB agar plates. vironmental sample in order to modulate the microbial community with respect to function (in situ or ex situ). Selection of microbial consortia degrading wheat straw The selective media that are used enhance the prevalence from three inoculum sources of desired functions in a microbial community and so the genes and/or operons of interest, as was shown for chitin The sources of the microbial communities used in this exper- (Cretoiu et al. 2012) as well as hemicellulose degradation iment were forest soil, decaying wood and canal sediment. processes (Jiménez et al. 2013). Forest soil encompassed three randomly collected (53.41 N; Microbial consortia with effective lignocellulose degrada- 6.90 E) 10-g surface (0–10 cm) samples which were thor- tion capacity can be selected from different source materials oughly mixed. The decaying wood was collected at the same (Wongwilaiwalin et al. 2010;Wangetal.2011; Jiménez et al. site. A 20-cm decomposing tree branch (hardwood), with ev- 2013;Moraïsetal.2014), leading to effective and structurally- ident microbial growth on its surface, was used. In the labo- stable consortia that successfully degrade substrates even be- ratory, it was cut into small (<3 mm) pieces in sterile condi- yond the ones they were selected upon (Haruta et al. 2002). tions. Sediment was collected from the bottom of an adjacent However, little is known about the differences that might arise canal, using three random samples of about 10 g, which were when different microbial sources are used to breed such de- thoroughly mixed. All samples were taken in February 2014 grader consortia on the same substrate, i.e., whether microbial (winter season). Cell suspensions were prepared by adding communities originating from different sources would con- 10 g of each of the microbial sources to 250 mL flasks con- verge to similar consortia when exposed to enrichment cultur-