Clouds constitute the uppermost layer of the biosphere. They host diverse communities whose functioning remains obscure, although biological activity potentially participates to atmospheric chemical ...and physical processes. In order to gain information on the metabolic functioning of microbial communities in clouds, we conducted coordinated metagenomics/metatranscriptomics profiling of cloud water microbial communities. Samples were collected from a high altitude atmospheric station in France and examined for biological content after untargeted amplification of nucleic acids. Living microorganisms, essentially bacteria, maintained transcriptional and translational activities and expressed many known complementary physiological responses intended to fight oxidants, osmotic variations and cold. These included activities of oxidant detoxification and regulation, synthesis of osmoprotectants/cryoprotectants, modifications of membranes, iron uptake. Consistently these energy-demanding processes were fueled by central metabolic routes involved in oxidative stress response and redox homeostasis management, such as pentose phosphate and glyoxylate pathways. Elevated binding and transmembrane ion transports demonstrated important interactions between cells and their cloud droplet chemical environments. In addition, polysaccharides, potentially beneficial for survival like exopolysaccharides, biosurfactants and adhesins, were synthesized. Our results support a biological influence on cloud physical and chemical processes, acting notably on the oxidant capacity, iron speciation and availability, amino-acids distribution and carbon and nitrogen fates.
Clouds are key components in Earth's functioning. In addition of acting as obstacles to light radiations and chemical reactors, they are possible atmospheric oases for airborne microorganisms, ...providing water, nutrients and paths to the ground. Microbial activity was previously detected in clouds, but the microbial community that is active in situ remains unknown. Here, microbial communities in cloud water collected at puy de Dôme Mountain's meteorological station (1465 m altitude, France) were fixed upon sampling and examined by high-throughput sequencing from DNA and RNA extracts, so as to identify active species among community members. Communities consisted of ~103-104 bacteria and archaea mL-1 and ~102-103 eukaryote cells mL-1. They appeared extremely rich, with more than 28 000 distinct species detected in bacteria and 2 600 in eukaryotes. Proteobacteria and Bacteroidetes largely dominated in bacteria, while eukaryotes were essentially distributed among Fungi, Stramenopiles and Alveolata. Within these complex communities, the active members of cloud microbiota were identified as Alpha- (Sphingomonadales, Rhodospirillales and Rhizobiales), Beta- (Burkholderiales) and Gamma-Proteobacteria (Pseudomonadales). These groups of bacteria usually classified as epiphytic are probably the best candidates for interfering with abiotic chemical processes in clouds, and the most prone to successful aerial dispersion.
The microorganisms living on the phyllosphere (the aerial part of the plants) are in contact with the lignocellulosic plant cell wall and might have a lignocellulolytic potential. We isolated a
...Saccharibacillus
strain (
Saccharibacillus
WB17) from wheat bran phyllosphere and its cellulolytic and hemicellulolytic potential was investigated during growth onto wheat bran. Five other type strains from that genus selected from databases were also cultivated onto wheat bran and glucose. Studying the chemical composition of wheat bran residues by FTIR after growth of the six strains showed an important attack of the stretching C-O vibrations assigned to polysaccharides for all the strains, whereas the C = O bond/esterified carboxyl groups were not impacted. The genomic content of the strains showed that they harbored several CAZymes (comprised between 196 and 276) and possessed four of the fifth modules reflecting the presence of a high diversity of enzymes families. Xylanase and amylase activities were the most active enzymes with values reaching more than 4746 ± 1400 mIU/mg protein for the xylanase activity in case of
Saccharibacillus deserti
KCTC 33693
T
and 452 ± 110 mIU/mg protein for the amylase activity of
Saccharibacillus
WB17. The total enzymatic activities obtained was not correlated to the total abundance of CAZyme along that genus. The
Saccharibacillus
strains harbor also some promising proteins in the GH30 and GH109 modules with potential arabinofuranosidase and oxidoreductase activities. Overall, the genus
Saccharibacillus
and more specifically the
Saccharibacillus
WB17 strain represent biological tools of interest for further biotechnological applications.
Purpose
Microbial fermentation on agro-industrial co-products is an interesting strategy for the bioproduction of metabolites of interest, as it lowers the costs of production and uses renewable and ...abundant carbon sources. Violacein is a purple pigment with interesting properties that is commonly produced by fermentation.
Chromobacterium vaccinii
, a wild-type, natural and non-toxic Proteobacteria (Pseudomonadota), has the ability to produce violacein. However, to select an optimal co-product as carbon source, it is necessary to understand its use of the different constituents of plant biomass.
Methods
Growth and violacein production of
C
.
vaccinii
were first studied on the main components of plant biomass. Then, they were assessed in presence of raw co-products at various concentrations. Tryptophan, a precursor of violacein biosynthesis, was added in order to evaluate the impact on violacein production yields.
Results
C
.
vaccinii
was able to grow on gluten (wheat protein), as well as on low concentrations of glucose. Selecting protein-rich substrates such as soybean and rapeseed cakes led to improved growth and violacein bioproduction. Addition of tryptophan led to net increases in violacein, but had low impacts on the bacterial growth.
Conclusion
Understanding microbial growth mechanisms during bioproduction of molecules of interest is key in order to select the best adapted agro-industrial co-products used as substrate. This study allowed to better characterize growth of
C
.
vaccinii
on various carbon sources and plant biomass, and showed that an optimal substrate with the addition of tryptophan could increase greatly the bioproduction yields of violacein by
C
.
vaccinii
.
Graphical abstract
Summary
Chloromethane (CH3Cl) is the most abundant halogenated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3Cl has mainly natural sources such as ...emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. We identified and characterized CH3Cl‐degrading bacteria associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13C‐labelled CH3Cl combined with metagenomics. Metagenome‐assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterized methylotrophic cmuA‐independent pathway may drive CH3Cl degradation in the investigated tree ferns.
•An easy set-up of the co-cultures from 2 different microorganisms (filamentous fungi and bacteria) from different microbial domains resulting into a greater and more diverse metabolic and ...lignocellulolytic content.•An over expression of several key enzymatic lignocellulolytic activities is observed during the co-coculture due to elicitation.•An elicitation of some specific biosynthetic cluster genes is observed due to the activation of those the complexity of the carbon compounds present in the lignocellulose.•An elicitation of some specific biosynthetic cluster genes is observed only during the co-culture experiment.•A specific microbial crosstalk and interaction exists at the species level between the 3 Streptomyces and the fungi leading to a specific of lignocellulolytic enzyme and secondary metabolite production.
Lignocellulose, the most abundant biomass on Earth, is a complex recalcitrant material mainly composed of three fractions: cellulose, hemicelluloses and lignins. In nature, lignocellulose is efficiently degraded for carbon recycling. Lignocellulose degradation involves numerous microorganisms and their secreted enzymes that act in synergy. Even they are efficient, the natural processes for lignocellulose degradation are slow (weeks to months). In this study, the objective was to study the synergism of some microorganisms to achieve efficient and rapid lignocellulose degradation. Wheat bran, an abundant co-product from milling industry, was selected as lignocellulosic biomass. Mono-cultures and co-cultures involving one A.niger strain fungi never sequenced before (DSM 1957) and either one of three different Streptomyces strains were tested in order to investigate the potentiality for efficient lignocellulose degradability. Comparative genomics of the strain Aspergillus niger DSM 1957 revealed that it harboured the maximum of AA, CBM, CE and GH among its closest relative strains. The different co-cultures set-up enriched the metabolic diversity and the lignocellulolytic CAZyme content. Depending on the co-cultures, an over-expression of some enzymatic activities (xylanase, glucosidase, arabinosidase) was observed in the co-cultures compared to the mono-cultures suggesting a specific microbial cross-talk depending on the microbial partner. Moreover, metabolomics for each mono and co-culture was performed and revealed an elicitation of the production of secondary metabolites and the activation of silent biosynthetic cluster genes depending on the microbial co-culture. This opens opportunities for the bioproduction of molecules of interest from wheat bran.
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Even though the 16S rRNA gene is the most commonly used taxonomic marker in microbial ecology, its poor resolution is still not fully understood at the intra-genus level. In this work, the number of ...rRNA gene operons, intra-genomic heterogeneities and lateral transfers were investigated at a fine-scale resolution, throughout the Pseudomonas genus. In addition to nineteen sequenced Pseudomonas strains, we determined the 16S rRNA copy number in four other Pseudomonas strains by Southern hybridization and Pulsed-Field Gel Electrophoresis, and studied the intra-genomic heterogeneities by Denaturing Gradient Gel Electrophoresis and sequencing. Although the variable copy number (from four to seven) seems to be correlated with the evolutionary distance, some close strains in the P. fluorescens lineage showed a different number of 16S rRNA genes, whereas all the strains in the P. aeruginosa lineage displayed the same number of genes (four copies). Further study of the intra-genomic heterogeneities revealed that most of the Pseudomonas strains (15 out of 19 strains) had at least two different 16S rRNA alleles. A great difference (5 or 19 nucleotides, essentially grouped near the V1 hypervariable region) was observed only in two sequenced strains. In one of our strains studied (MFY30 strain), we found a difference of 12 nucleotides (grouped in the V3 hypervariable region) between copies of the 16S rRNA gene. Finally, occurrence of partial lateral transfers of the 16S rRNA gene was further investigated in 1803 full-length sequences of Pseudomonas available in the databases. Remarkably, we found that the two most variable regions (the V1 and V3 hypervariable regions) had probably been laterally transferred from another evolutionary distant Pseudomonas strain for at least 48.3 and 41.6% of the 16S rRNA sequences, respectively. In conclusion, we strongly recommend removing these regions of the 16S rRNA gene during the intra-genus diversity studies.
Purpose
The continuing search for more efficient, low-cost enzymes for biomass hydrolysis is a crucial issue in plant biorefining. Due to their ability to secrete a broad set of plant cell-wall ...degrading enzymes, phytopathogenic wood-colonizing fungi depict a promising source of lignocellulolytic enzymes. However, the use of phytopathogenic fungal enzymes in plant biorefining remains poorly explored. Here, we assessed the capacity of lignocellulolytic enzymes produced by the grapevine trunk pathogen
Neofusicoccum parvum
in the breakdown of non-pretreated wheat straw (WS) and grapevine canes (GP) after growth on these two substrates.
Methods
First, we determined the peak activity time of lignocellulases during 42 days of cultivation. In addition, transcriptomics at late growth stages was performed. Second, we evaluated the performance of secreted enzymes from the WS (WS-S) and GP (GP-S) cultures in hydrolyzing both lignocellulose substrates.
Results
Most of the enzymes reached the maximum activity between 14 and 28 days post-inoculation. Enzyme assays combined with transcriptomics revealed a dynamic enzyme utilization, where (hemi-)cellulases were predominantly produced at early growth stages. Afterward, enzymes acting on more recalcitrant polymers became more prominent. The WS-S released more xylose, arabinose, and total sugars from WS (3.3, 7.4, and 4.9% yields, respectively) than GP-S, although the latter generated more glucose from GP (2.4% yield). The sugar yields produced by
N. parvum
enzymes, particularly on WS hydrolysis, were comparable with those reported for typical industrial-use fungi, such as
Aspergillus
and
Trichoderma
spp.
Conclusion
These findings suggest that
N. parvum
may be a helpful source of lignocellulolytic enzymes for plant biorefining.
Statement of Novelty
The efficient enzymatic conversion of lignocellulose to fermentable sugars is one of the major bottlenecks in plant biorefining. That has led to a constant search for novel lignocellulolytic enzymes. Plant pathogens represent a rich but little exploited source of cell wall-degrading enzymes. This work shows that lignocellulolytic enzymes from the fungal trunk pathogen
Neofusicoccum parvum
performed comparatively well in converting non-pretreated wheat straw and grapevine canes into monomeric sugars. Our results suggest that both agricultural residues can be used as a cheap carbon source for producing enzymes of interest for plant biorefining, which may contribute to their valorization, particularly for the less explored grapevine pruning residues. In addition, we described a temporal utilization of lignocellulolytic enzyme in
N. parvum
.
Highlights
Temporal dynamics in lignocellulolytic enzyme activities from
N. parvum
.
Up-regulation of CAZymes-coding genes after long-term in vitro cultivation.
N. parvum
enzymes seem promising for hydrolysis of non-pretreated lignocellulose.
Graphical Abstract
Chloromethane (CH3Cl) is the most abundant halogenated trace gas in the atmosphere. It plays an important role in natural stratospheric ozone destruction. Current estimates of the global CH3Cl budget ...are approximate. The strength of the CH3Cl global sink by microbial degradation in soils and plants is under discussion. Some plants, particularly ferns, have been identified as substantial emitters of CH3Cl. Their ability to degrade CH3Cl remains uncertain. In this study, we investigated the potential of leaves from 3 abundant ferns (Osmunda regalis, Cyathea cooperi, Dryopteris filix-mas) to produce and degrade CH3Cl by measuring their production and consumption rates and their stable carbon and hydrogen isotope signatures. Investigated ferns are able to degrade CH3Cl at rates from 2.1 to 17 and 0.3 to 0.9μggdw−1day−1 for C. cooperi and D. filix-mas respectively, depending on CH3Cl supplementation and temperature. The stable carbon isotope enrichment factor of remaining CH3Cl was −39±13‰, whereas negligible isotope fractionation was observed for hydrogen (−8±19‰). In contrast, O. regalis did not consume CH3Cl, but produced it at rates ranging from 0.6 to 128μggdw−1day−1, with stable isotope values of −97±8‰ for carbon and −202±10‰ for hydrogen, respectively. Even though the 3 ferns showed clearly different formation and consumption patterns, their leaf-associated bacterial diversity was not notably different. Moreover, we did not detect genes associated with the only known chloromethane utilization pathway “cmu” in the microbial phyllosphere of the investigated ferns. Our study suggests that still unknown CH3Cl biodegradation processes on plants play an important role in global cycling of atmospheric CH3Cl.
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•Ferns both produce and degrade atmospheric CH3Cl with large individual variations.•Ferns degrade CH3Cl at rates ranging from 0.3 to 17μg·g(dry weight)−1day−1.•CH3Cl degradation was correlated to a large εC and almost no εH isotope effect.•Involvement of the bacterial cmu pathway in CH3Cl degradation was not detected.•Still unknown CH3Cl biodegradation processes in plants contribute to the CH3Cl cycle.
The
Acidobacteria
phylum is a very abundant group (20–30% of microbial communities in soil ecosystems); however, little is known about these microorganisms and their ability to degrade the biomass ...and lignocellulose due to the difficulty of culturing them. We, therefore, bioinformatically studied the content of lignocellulolytic enzymes (total and predicted secreted enzymes) and secreted peptidases in an in silico library containing 41
Acidobacteria
genomes. The results showed a high abundance and diversity of total and secreted Carbohydrate-Active enzymes (cazyme) families among the
Acidobacteria
compared to known previous degraders. Indeed, the relative abundance of cazymes in some genomes represented more than 6% of the gene coding proteins with at least 300 cazymes. The same observation was made with the predicted secreted peptidases with several families of secreted peptidases, which represented at least 1.5% of the gene coding proteins in several genomes. These results allowed us to highlight the lignocellulolytic potential of the
Acidobacteria
phylum in the degradation of lignocellulosic biomass, which could explain its high abundance in the environment.
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