Dynamics of Abundant and Rare Bacteria During Degradation of Lignocellulose from Sugarcane Biomass.

Microorganisms play a crucial role in lignocellulosic degradation. Many enriched microbial communities have demonstrated to reach functional and structural stability with effective degrading capacities of industrial interest. These microbial communities are typically composed by only few dominant species and a high number of usually overlooked rare species. Here, we used two sources of lignocellulose (sugarcane bagasse and straw) in order to obtain lignocellulose-degrading bacteria through an enriched process, followed the selective trajectory of both abundant and rare bacterial communities by 16S rRNA gene amplification and analyzed the outcomes of selection in terms of capacities and specialization. We verified the importance of pre-selection by using two sources of microbial inoculum: soil samples from a sugarcane field with history of straw addition (St15) and control samples, from the same field, without amendments (St0). We found similitudes in terms of stabilization between the abundant and rare fractions. We also found positive correlations of both abundant and rare taxa (like Caulobacteraceae and Alcaligenaceae) and the degradation of lignocellulosic fractions. Differences in the inocula's initial diversity rapidly decreased during the enrichment resulting in comparable richness levels at the end of the process; however, the legacy of the St15 inoculum and its specialization positively influenced the degradation capacities of the community. Analysis of specialization of the final communities revealed increased straw degradation capacity in the communities enriched in bagasse, which could be potentially used as a strategy for improving lignocellulose waste degradation on the sugarcane fields. This work highlights the importance of including the rare fraction of bacterial communities during investigations involving the screening and assessment of effective degrading communities.

Construction of Effective Minimal Active Microbial Consortia for Lignocellulose Degradation.

Enriched microbial communities, obtained from environmental samples through selective processes, can effectively contribute to lignocellulose degradation. Unfortunately, fully controlled industrial degradation processes are difficult to reach given the intrinsically dynamic nature and complexity of the microbial communities, composed of a large number of culturable and unculturable species. The use of less complex but equally effective microbial consortia could improve their applications by allowing for more controlled industrial processes. Here, we combined ecological theory and enrichment principles to develop an effective lignocellulose-degrading minimal active microbial Consortia (MAMC). Following an enrichment of soil bacteria capable of degrading lignocellulose material from sugarcane origin, we applied a reductive-screening approach based on molecular phenotyping, identification, and metabolic characterization to obtain a selection of 18 lignocellulose-degrading strains representing four metabolic functional groups. We then generated 65 compositional replicates of MAMC containing five species each, which vary in the number of functional groups, metabolic potential, and degradation capacity. The characterization of the MAMC according to their degradation capacities and functional diversity measurements revealed that functional diversity positively correlated with the degradation of the most complex lignocellulosic fraction (lignin), indicating the importance of metabolic complementarity, whereas cellulose and hemicellulose degradation were either negatively or not affected by functional diversity. The screening method described here successfully led to the selection of effective MAMC, whose degradation potential reached up 96.5% of the degradation rates when all 18 species were present. A total of seven assembled synthetic communities were identified as the most effective MAMC. A consortium containing Stenotrophomonas maltophilia, Paenibacillus sp., Microbacterium sp., Chryseobacterium taiwanense, and Brevundimonas sp. was found to be the most effective degrading synthetic community.

Differential stress resistance and metabolic traits underlie coexistence in a sympatrically evolved bacterial population.

Following intermittent batch growth in Luria-Bertani (LB) broth for about 1000 generations, differentially evolved forms were found in a population of Escherichia coli cells. Studies on this population revealed the emergence of key polymorphisms, as evidenced by analysis of both whole genome sequences and transcription analysis. Here, we investigated the phenotypic nature of several key forms and found a remarkable (interactive) coexistence of forms which highlights the presence of different ecological roles pointing at a dichotomy in: (i) tolerance to environmental stresses and (ii) the capacity to utilize particular carbon sources such as galactose. Both forms differed from their common ancestor by different criteria. This apparent coexistence of two diverged forms points at the occurrence of niche partitioning as a consequence of dichotomous adaptive evolution. Remarkably, the two forms were shown to continue to coexist - in varying ratio's - in an experiment that cycled them through periods of nutrient feast (plentiful growth substrates) and famine (growth-restrictive - stress conditions). The results further indicated that the equilibrium of the coexistence was destroyed when one of the parameters was high tuned, jeopardizing the stability of the coexisting pair.

Sympatric metabolic diversification of experimentally evolved Escherichia coli in a complex environment.

Sympatric diversification in bacteria has been found to contravene initial evolutionary theories affirming the selection of the fittest type by competition for the same resource. Studies in unstructured (well-mixed) environments have discovered divergence of an ancestor strain into genomically and phenotypically divergent types growing both on single and mixed energy sources. This study addresses the metabolic diversification in an Escherichia coli population that evolved over ~1,000 generations under aerobic conditions in the nutritional complexity offered by Luria-Bertani (LB) broth. The medium lacked glucose but contained a variety of other resources. Two distinct metabolically-diverged types, coinciding with colony morphologies, were found to dominate the populations. One type was an avid carbohydrate consumer, which could quickly utilize the available (alternative) substrates feeding into glycolysis. The second type was a slow grower, which was able to specifically consume acetate. The capacity to utilize acetate might be providing an advantage to this second type, suggesting an increased capability to deal with adverse conditions that occur in the later stages of growth. The diverged metabolic preferences of the two forms suggested differential and interactive ecological roles within the population. We postulate that these types used different alternative metabolic strategies occupying different niches in a sympatric manner as an outcome of adaptation to the complex environment.

Comparative genomics and transcriptomics analysis of experimentally evolved Escherichia coli MC1000 in complex environments.

It has recently become feasible to study the basis and nature of evolutionary changes in bacteria in an experimental setting using defined media. However, assessment of adaptive changes in complex environments has been scarce. In an effort to describe the responses in such environments, we unravel, in a comparative approach, the transcriptional and genetic profiles of 19 Escherichia coli strains that evolved in Luria Bertani medium under three different oxygen regimes over 1000 generations. A positive relationship between upregulation of gene expression and the number of mutations was observed, suggesting that a number of metabolic pathways were activated. Phenotypic polymorphisms were observed in parallel cultures, of which some were related with mutations at the regulatory level. Non-parallel responses were observed at the intrapopulational level, which is indicative of diversifying selection. Parallel responses encompassed transcriptome diversity, and their effects were directly affected by differing genomic backgrounds. A fluctuating selective force produced higher phenotypic diversity compared with constant forces. This study demonstrates how phenotypic innovations may depend on the relationship between genomic changes and local ecological conditions. Using both comparative genomics and transcriptomics approaches, the results help elucidating various adaptive responses in cultures in unexplored complex environments.

Adaptation and heterogeneity of Escherichia coli MC1000 growing in complex environments.

In a study aiming to assess bacterial evolution in complex growth media, we evaluated the long-term adaptive response of Escherichia coli MC1000 in Luria-Bertani (LB) medium. Seven parallel populations were founded and followed over 150 days in sequential batch cultures under three different oxygen conditions (defined environments), and 19 evolved forms were isolated. The emergence of forms with enhanced fitness was evident in competition experiments of all evolved forms versus the ancestral strain. The evolved forms were then subjected to phenotypic and genomic analyses relative to the ancestor. Profound changes were found in their phenotypes as well as whole-genome sequences. Interestingly, considerable heterogeneity was found at the intrapopulational level. However, consistently occurring parallel adaptive responses were found across all populations. The evolved forms all contained a mutation in galR, a repressor of the galactose operon. Concomitantly, the new forms revealed enhanced growth on galactose as well as galactose-containing disaccharides. This response was likely driven by the LB medium.

Efecto de diferentes métodos de cultivo sobre la regeneración in vitro de papa (Solanum tuberosum L.) de la vaeriedad comerciall Chieftain

Se evaluó la capacidad de regeneración de explantes de hoja, tubérculos y segmentos de tallo de la variedad comercial Chieftan, utilizando dos métodos: A, el mismo medio de cultivo desde la iniciación de los callos hasta la formación de los brotes y B, transferencia de los callos a un medio de cultivo libre de auxinas antes de la formación de los brotes. La mayor frecuencia de regeneración se obtuvo de discos de tubérculo en el medio (MRT-6) (método A) con 3,28 brotes por explante, seguido por 2,5 brotes en segmentos de tallo (medios MRE-4 y MRE-6, método B) y 0,8 brotes a partir de discos de hoja (medio MRH-3, método A). La combinación de 1 mg/L de nitrato de plata con 5 g/L de carbón activado, durante la propagación in vitro, mostró diferencias significativas tanto en el número de hojas por planta como en su calidad (AU)