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Is a floodplain forest a valuable source for lignin-degrading anaerobic microbial communities: A metagenomic approach
Abstract
Lignin is one of the most substantial obstacles in the evaluation of lignocellulosic compounds. Although there are numerous approaches for the enhancement of lignin digestion in the literature, there has yet to be an optimized system to date. In this study, samples taken from Igneada floodplain forests were enriched anaerobically at 25 °C and 37 °C, with alkali lignin as the sole carbon source. The activity of the anaerobic lignin-degrading microbial consortium was detected more efficiently at 37 °C, where biogas production exceeded 3.5 mLgas/mLmedium. It was observed that the microbial community initially dominated by Proteobacteria (around 60%) changed completely after enrichment and was led by members of the Firmicutes phylum (up to 90%). The dominant species (Sporomusa termitida, Desulfitobacterium hafniense, Citrobacter freundii, Citrobacter portucalensis, Alkalibacter rhizosphaerae, and Gudongella oleilytica) occupying more than 50% in the final enrichment culture were only around 2% in the raw samples. Therefore, this study, one of the few in which enriched environmental samples were sequenced using MinION, demonstrated that longoses are exceptional reservoirs for lignin-digesting anaerobic microorganisms.
... Despite the considerable challenges associated with anaerobic degradation of lignin, even deemed infeasible, a recent paper has provided evidence to the contrary (Lankiewicz et al., 2023). Furthermore, recent studies have demonstrated the presence of certain species and microbial consortia capable of anaerobically degrading lignin (Billings et al., 2015;DeAngelis et al., 2011;Ozsefil et al., 2023;Woo et al., 2014). Thus, while anaerobic degradation of lignin appears feasible, enhancing lignin degradation efficiency in anaerobic digestion systems remains crucial. ...
... However, the latter is considered to be more effective due to the capacity of initiating potential synergistic relationship between microorganisms as well as adaptation to environmental conditions (Tsegaye et al., 2019). Therefore, the enriched lignin-degrading anaerobic microbial community from floodplain forests, obtained in our previous study (Ozsefil et al., 2023), is highly suitable for development of such a microbial consortium mentioned above. While there are studies utilizing lignin-rich lignocellulosic substrates, there is currently a gap in the literature concerning bioaugmentation studies specifically employing lignin as the sole substrate or investigating microbial consortia that anaerobically degrade lignin, to the best of our knowledge. ...
... This enrichment process was conducted at two conditions: 25 • C, which approximates the ambient temperature of the habitat where the samples were collected, and 37 • C, an ideal temperature for industrial applications. The enrichment was carried out in an oxygen-free environment, as detailed in our previous study (Ozsefil et al., 2023). After the cultivation process, enrichment cultures were transferred to the batch digesters and subjected to BMP assay. ...
A B S T R A C T
The recalcitrance of lignin impedes the efficient utilization of lignocellulosic biomass, hindering the efficient
production of biogas and value-added materials. Despite the emergence of anaerobic digestion as a superior
alternative to the aerobic method for lignin processing, achieving its feasibility requires thorough characterization
of lignin-degrading anaerobic microorganisms, assessment of their biomethane production potential, and
a comprehensive understanding of the degradation pathway. This study aimed to address the aforementioned
necessities by bioaugmenting seed sludge with three distinct enriched lignin-degrading microbial consortia at
both 25 ◦C and 37 ◦C. Enhanced biomethane yields was detected in the bioaugmented digesters, while the
highest production was observed as 188 mLN CH4 gVS 1 in digesters operated at 37 ◦C. Moreover, methane yield
showed a significant improvement in the samples at 37 ◦C ranging from 110% to 141% compared to the control,
demonstrating the efficiency of the enriched lignin-degrading microbial community. Temperature and substrate
were identified as key factors influencing microbial community dynamics. The observation that microbial
communities tended to revert to the initial state after lignin depletion, indicating the stability of the overall
microbiota composition in the digesters, is a promising finding for large-scale studies. Noteworthy candidates for
lignin degradation, including Sporosarcina psychrophila, Comamonas aquatica, Shewanella baltica, Pseudomonas sp.
... Despite the considerable challenges associated with anaerobic degradation of lignin, even deemed infeasible, a recent paper has provided evidence to the contrary (Lankiewicz et al., 2023). Furthermore, recent studies have demonstrated the presence of certain species and microbial consortia capable of anaerobically degrading lignin (Billings et al., 2015;DeAngelis et al., 2011;Ozsefil et al., 2023;Woo et al., 2014). Thus, while anaerobic degradation of lignin appears feasible, enhancing lignin degradation efficiency in anaerobic digestion systems remains crucial. ...
... However, the latter is considered to be more effective due to the capacity of initiating potential synergistic relationship between microorganisms as well as adaptation to environmental conditions (Tsegaye et al., 2019). Therefore, the enriched lignin-degrading anaerobic microbial community from floodplain forests, obtained in our previous study (Ozsefil et al., 2023), is highly suitable for development of such a microbial consortium mentioned above. While there are studies utilizing lignin-rich lignocellulosic substrates, there is currently a gap in the literature concerning bioaugmentation studies specifically employing lignin as the sole substrate or investigating microbial consortia that anaerobically degrade lignin, to the best of our knowledge. ...
... This enrichment process was conducted at two conditions: 25 • C, which approximates the ambient temperature of the habitat where the samples were collected, and 37 • C, an ideal temperature for industrial applications. The enrichment was carried out in an oxygen-free environment, as detailed in our previous study (Ozsefil et al., 2023). After the cultivation process, enrichment cultures were transferred to the batch digesters and subjected to BMP assay. ...
... Despite the considerable challenges associated with anaerobic degradation of lignin, even deemed infeasible, a recent paper has provided evidence to the contrary (Lankiewicz et al., 2023). Furthermore, recent studies have demonstrated the presence of certain species and microbial consortia capable of anaerobically degrading lignin (Billings et al., 2015;DeAngelis et al., 2011;Ozsefil et al., 2023;Woo et al., 2014). Thus, while anaerobic degradation of lignin appears feasible, enhancing lignin degradation efficiency in anaerobic digestion systems remains crucial. ...
... However, the latter is considered to be more effective due to the capacity of initiating potential synergistic relationship between microorganisms as well as adaptation to environmental conditions (Tsegaye et al., 2019). Therefore, the enriched lignin-degrading anaerobic microbial community from floodplain forests, obtained in our previous study (Ozsefil et al., 2023), is highly suitable for development of such a microbial consortium mentioned above. While there are studies utilizing lignin-rich lignocellulosic substrates, there is currently a gap in the literature concerning bioaugmentation studies specifically employing lignin as the sole substrate or investigating microbial consortia that anaerobically degrade lignin, to the best of our knowledge. ...
... This enrichment process was conducted at two conditions: 25 • C, which approximates the ambient temperature of the habitat where the samples were collected, and 37 • C, an ideal temperature for industrial applications. The enrichment was carried out in an oxygen-free environment, as detailed in our previous study (Ozsefil et al., 2023). After the cultivation process, enrichment cultures were transferred to the batch digesters and subjected to BMP assay. ...
... For VFA analysis, samples were centrifuged at 14.000 rpm at 4 °C for 30 min, and supernatants were collected and filtered through 0.22-µm pore size membrane filters before VFA measurements. Afterward, 10 N phosphoric acid was added to the final filtrates of the samples at a concentration of 10% by volume [30]. VFAs were measured by a gas chromatograph (GC-2025, Shimadzu Co., Japan) equipped with an auto injector (AOC-20i, Shimadzu Co., Japan). ...
... For SS, DS, and DST, the values in both analyses remained stable or increased after 30 days of incubation (Fig. 3b, c). However, a different trend was observed for sample D. It can be inferred that species diversity and abundance decreased in the case of sample D (p = 0.02), likely due to the stress induced by the substrate [30]. This phenomenon is commonly referred to as the microcosm effect in the literature [52]. ...
The valorization of food processing waste is critical for sustainable development and circular economy frameworks. Although Turkish delight waste (D), a high-volume byproduct of the confectionery industry in Turkey, holds significant potential for valorization within the circular economy framework, this potential remains largely unexplored. This study evaluated the valorization potential of Turkish delight waste in anaerobic digester systems by co-digesting it with sunflower heads (S) and tea stalks (T), common agricultural wastes in Turkey, in addition to mono-digestion. Economic evaluations were also conducted on the products obtained at different stages of anaerobic digestion (AD). The highest methane yield of 388 mL CH4/g VS was obtained from Turkish delight waste in mono-digestion, and this substrate enhanced methane production when co-digested with the other substrates. However, co-digestion in the DST digester combining all three substrates yielded 234 mL CH4/g VS, indicating a limited synergistic effect. Metagenomic analyses revealed substrate-dependent variations in microbial community dynamics, particularly in digesters containing only Turkish delight waste. Acidogenic fermentation aimed at increasing VFA yield resulted in total acetic acid productions of 2828, 1707, and 1261 mg/L for D, DS, and DST, respectively. Economic assessments demonstrated that the value derived from VFA production was nearly double that obtained from methane production, even in cases where co-digestion resulted in lower overall yields. Thus, Turkish delight waste emerges as a promising candidate for both mono- and co-digestion in AD systems, offering a potentially more economically viable alternative to methane production through the generation of value-added chemicals such as VFAs.
Graphical Abstract
... Phylogenetic analyses, alpha and beta diversity assessments, PCA, PCoA, biomarker, phenotype analyses, and dynamic Krono charts were executed using Qiime2 (v.2019.7) (https://qiime2.org/)[17], while Mothur (v.1.48.0) organized taxonomic classifications (https://mothur.org/wiki/miseq_sop/). ...
Microplastics (MPs) are major concern due to their potential harm to ecosystems and most research has focused on their presence and fate, with limited attention to their biodegradation in aquatic ecosystems. Nevertheless, MPs act as hotspots for the colonization by a diverse range of microorganisms that can adhere to plastic surfaces, resulting in the subsequent formation of biofilms—a potential threat especially in terms of pathogenicity. This study employed 16S rRNA and 18S rRNA sequencing metagenomic analyses to investigate microbial communities within biofilms on plastic materials exposed to long‐term marine and freshwater environments. Three Arcobacter species (Arcobacter nitrofigilis, Arcobacter acticola, and Arcobacter suis) emerged as dominant species in M_MP sample, while Flavobacterium tructae was the predominant species within the F_MP sample. The 18S rRNA sequencing revealed the presence of the fungal phylum Ascomycota and the microalgal species Pseudocharaciopsis ovalis in F_MP. Although, the primary species detected on M_MP and F_MP samples include bacteria previously implicated as pathogen, the predominant species identified in this study were unconnected to MP‐associated biofilms or MP degradation. Their presence constitutes a novel discovery, opening promising avenues for the exploration of their potential involvement in the biodegradation of MPs within aquatic environments.
The Pachnoda marginata larva have complex gut microbiota capable of the effective conversion of lignocellulosic biomass. Biotechnological utilization of these microorganisms in an engineered system can be achieved by establishing enrichment cultures using a lignocellulosic substrate. We established enrichment cultures from contents of the midgut and hindgut of the beetle larva using wheat straw in an alkaline medium at mesophilic conditions. Two different inoculation preparations were used: procedure 1 (P1) was performed in a sterile bench under oxic conditions using 0.4% inoculum and small gauge needles. Procedure 2 (P2) was carried out under anoxic conditions using more inoculum (4%) and bigger gauge needles. Higher methane production was achieved with P2, while the highest acetic acid concentrations were observed with P1. In the enrichment cultures, the most abundant bacterial families were Dysgonomonadaceae, Heliobacteriaceae, Ruminococcaceae, and Marinilabiliaceae. Further, the most abundant methanogenic genera were Methanobrevibacter, Methanoculleus, and Methanosarcina. Our observations suggest that in samples processed with P1, the volatile fatty acids were not completely converted to methane. This is supported by the finding that enrichment cultures obtained with P2 included acetoclastic methanogens, which might have prevented the accumulation of acetic acid. We conclude that differences in the inoculum preparation may have a major influence on the outcome of enrichment cultures from the P. marginata larvae gut.
Genome reconstruction from metagenomes enables detailed study of individual community members, their metabolisms, and their survival strategies. Obtaining high quality metagenome-assembled genomes (MAGs) is particularly valuable in extreme environments like sea ice cryoconites, where the native consortia are recalcitrant to culture and strong astrobiology analogues. We evaluated three separate approaches for MAG generation from Allen Bay, Nunavut sea ice cryoconites—HiSeq-only, MinION-only, and hybrid (HiSeq + MinION)—where field MinION sequencing yielded a reliable metagenome. The hybrid assembly produced longer contigs, more coding sequences, and more total MAGs, revealing a microbial community dominated by Bacteroidetes. The hybrid MAGs also had the highest completeness, lowest contamination, and highest N50. A putatively novel species of Octadecabacter is among the hybrid MAGs produced, containing the genus’s only known instances of genomic potential for nitrate reduction, denitrification, sulfate reduction, and fermentation. This study shows that the inclusion of MinION reads in traditional short read datasets leads to higher quality metagenomes and MAGs for more accurate descriptions of novel microorganisms in this extreme, transient habitat and has produced the first hybrid MAGs from an extreme environment.
Biogas production from agricultural residues represents an effective and sustainable option for handling vast quantities of lignocellulosic waste for meeting the global energy demand. However, the recalcitrance of lignocellulosic biomass due to the presence of cellulose and hemicellulose in a structurally complex lignocellulosic matrix, and the crystallinity of cellulose create a hindrance in biogas production by restricting the availability of fermentable sugars to microbial action. This paper assesses the different pretreatment strategies adopted for making the lignocellulosic biomass amenable to anaerobic digestion by altering the structure of lignocellulose and eliminating lignin, thus increasing the accessibility of microbes to the easily degradable components. The review highlights the advantages and limitations of each technology—physical for size reduction (chipping, milling, extrusion, cavitation), thermal for breaking down of hydrogen bonds (conventional heating, steam explosion, microwave irradiation, hydrothermal), chemical for decreasing the crystallinity and polymerization degree of cellulose (alkalis, acids, gases, oxidizing agents, various solvents), biological for enhancing the digestibility (microbial, enzymatic), and their effect on improving the degradation efficiency and biogas yield. Further, the review discusses the role of some emerging technologies and other less explored options which may require optimization at higher scale so that the confidence in the translation of this knowledge can be increased and this abundantly available raw material can be effectively utilized. With the goal of improving the applicability of pretreatment technologies, some recommendations are put forth so that the efficiency of anaerobic digestion can be increased and the development of pretreatment technologies can be promoted on a large scale.
Huge quantum of agricultural crop residue from agrarian economies like India is disposed of improperly by open land burning contributing to particulate and gaseous pollutants thereby severely impacting the environment and human health. There is an overwhelming need for sustainable utilization of this misplaced resource as feedstock in biofuel production through anaerobic digestion. Substrate composition influences the digestibility of the substrate and underlying microbial plasticity involved in anaerobic digestion. In this study, anaerobic inoculum was subjected to enrichment in the presence of agricultural residues with different lignin content whose influence on biogas production, and differential modulation/ reorganization of microbial communities was explored. BMP assay emphasized the inverse proportionality between lignin content and biogas yield, with highest biogas yield of 622 mL/g VS added in case of rice straw (lowest lignin, 16.9%), followed by wheat straw—556 mL/g VS (18.12% lignin), sugar cane leaves—451 mL/g VS (21.87% lignin), and lowest biogas yield of 411 mL/g VS added in case of cotton crop waste (highest lignin, 23.97%). Biogas production efficiency was correlated with the selection and shift of microbial communities to key hemicellulolytic (Bacteroides, Clostridium, Prevotella, Thermotoga, Cytophaga) and lignocellulolytic (Bacillus, Geobacter, Flavobacterium, Spirochaeta, Klebsiella, Escherichia, Pseudomonas, Burkholderia) genotypes in comparison to initial anaerobic inoculum. Study highlighted that the variable lignin content resulted in genomic reorganization and created a scenario for high biogas production by selectively enriching different methanogenic pathways such as hydrogenotrophic pathways in rice straw metagenome (lowest lignin) and acetoclastic pathways in cotton crop waste metagenome (highest lignin).
High-throughput metagenomic sequencing is considered one of the main technologies fostering the development of microbial ecology. Widely used second-generation sequencers have enabled the analysis of extremely diverse microbial communities, the discovery of novel gene functions, and the comprehension of the metabolic interconnections established among microbial consortia. However, the high cost of the sequencers and the complexity of library preparation and sequencing protocols still hamper the application of metagenomic sequencing in a vast range of real-life applications. In this context, the emergence of portable, third-generation sequencers is becoming a popular alternative for the rapid analysis of microbial communities in particular scenarios, due to their low cost, simplicity of operation, and rapid yield of results. This review discusses the main applications of real-time, in situ metagenomic sequencing developed to date, highlighting the relevance of this technology in current challenges (such as the management of global pathogen outbreaks) and in the next future of industry and clinical diagnosis.
Volatile fatty acids (VFAs) has great potential for closed-loop production in dairy industries via resource recovery from waste-streams. In the current study, the transition of VFA production from the batch reactor to anaerobic sequencing batch reactor (ASBR) by using cheese industry wastewater under alkali pH was evaluated with respect to seed sludge structure, microbial diversity and reactor type. The transition from the batch reactor to ASBR demonstrated that the maximum VFA production yield (g COD/g SCOD) was comparable in two reactors (batch: 0.97; ASBR: 0.94), whereas, the dominant acid type was different (batch: 49% lactic acid; ASBR: 80% propionic acid). There was a significant correlation between the productions of butyric acid with Gracilibacteraceae and Desulfovibrionaceae; propionic acid with Desulfovibrionaceae and Synergistaceae; lactic acid with Pseudomonadaceae and Rhodocyclaceae. The high VFA production efficiency can be achieved by long term reactor operation, which enables the shift from industrial waste-streams to biorefineries.
Two bacterial strains, denoted so4 and w15, isolated from wheat straw (WS)-degrading microbial consortia, were found to grow synergistically in media containing WS as the single carbon and energy source. They were identified as Citrobacter freundii so4 and Sphingobacterium multivorum w15 based on 16S rRNA gene sequencing and comparison to the respective C. freundii and S. multivorum type strains. In order to identify the mechanisms driving the synergistic interactions, we analyzed the draft genomes of the two strains and further characterized their metabolic potential. The latter analyses revealed that the strains had largely complementary substrate utilization patterns, with only 22 out of 190 compounds shared. The analyses further indicated C. freundii so4 to primarily consume amino acids and simple sugars, with laminarin as a key exception. In contrast, S. multivorum w15 showed ample capacity to transform complex polysaccharides, including intermediates of starch degradation. Sequence analyses revealed C. freundii so4 to have a genome of 4,883,214 bp, with a G + C content of 52.5%, 4,554 protein-encoding genes and 86 RNA genes. S. multivorum w15 has a genome of 6,678,278 bp, with a G + C content of 39.7%, 5,999 protein-encoding genes and 76 RNA genes. Genes for motility apparatuses (flagella, chemotaxis) were present in the genome of C. freundii so4, but absent from that of S. multivorum w15. In the genome of S. multivorum w15, 348 genes had regions matching CAZy family enzymes and/or carbohydrate-binding modules (CBMs), with 193 glycosyl hydrolase (GH) and 50 CBM domains. Remarkably, 22 domains matched enzymes of glycoside hydrolase family GH43, suggesting a strong investment in the degradation of arabinoxylan. In contrast, 130 CAZy family genes were found in C. freundii so4, with 61 GH and 12 CBM domains identified. Collectively, our results, based on both metabolic potential and genome analyses, revealed the two strains to harbor complementary catabolic armories, with S. multivorum w15 primarily attacking the WS hemicellulose and C. freundii so4 the cellobiose derived from cellulose, next to emerging oligo- or monosaccharides. Finally, C. freundii so4 may secrete secondary metabolites that S. multivorum w15 can consume, and detoxify the system by reducing the levels of (toxic) by-products.
Clostridium sp. is a genus of anaerobic bacteria capable of metabolizing several substrates (monoglycerides, diglycerides, glycerol, carbon monoxide, cellulose, and more), into valuable products. Biofuels, such as ethanol and butanol, and several chemicals, such as acetone, 1,3-propanediol, and butyric acid, can be produced by these organisms through fermentation processes. Among the most well-known species, Clostridium carboxidivorans, C. ragsdalei, and C. ljungdahlii can be highlighted for their ability to use gaseous feedstocks (as syngas), obtained from the gasification or pyrolysis of waste material, to produce ethanol and butanol. C. beijerinckii is an important species for the production of isopropanol and butanol, with the advantage of using hydrolysate lignocellulosic material, which is produced in large amounts by first-generation ethanol industries. High yields of 1,3 propanediol by C. butyricum are reported with the use of another by-product from fuel industries, glycerol. In this context, several Clostridium wild species are good candidates to be used as biocatalysts in biochemical or hybrid processes. In this review, literature data showing the technical viability of these processes are presented, evidencing the opportunity to investigate them in a biorefinery context.
Biomass recalcitrance is still a main challenge for the production of biofuels and high‐value products. Here, an alternative Miscanthus pretreatment method by using lignin‐degrading bacteria was developed. Six efficient Miscanthus‐degrading bacteria were first cultured to produce laccase by using 0.5% Miscanthus biomass as carbon source. After 1–5 days of incubation, the maximum laccase activities induced by Miscanthus in the six strains were ranged from 103 to 8091 U l⁻¹. Then, the crude enzymes were directly diluted by equal volumes of citrate buffer and added Miscanthus biomass to a solid concentration at 4% (w/v). The results showed that all bacterial pretreatments significantly decreased the lignin content, especially in the presence of two laccase mediators (ABTS and HBT). The lignin removal directly correlated with increases in total sugar and glucose yields after enzymatic hydrolysis. When ABTS was used as a mediator, the best lignin‐degrading bacteria (Pseudomonas sp. AS1) can remove up to 50.1% lignin of Miscanthus by obtaining 2.2‐fold glucose yield, compared with that of untreated biomass. Therefore, this study provided an effective Miscanthus pretreatment method by using lignin‐degrading bacteria, which may be potentially used in improving enzymatic hydrolysability of biomass.
This study aims to test the spectral and spatial consistency of Sentinel-2 and Landsat-8 OLI data for the potential of monitoring longos forests for four seasons in Igneada, Turkey. Vegetation indices, including Normalized Difference Vegetation Index (NDVI), Enhanced Vegetation Index (EVI) and Normalized Difference Water Index (NDWI), were generated for the study area in addition to the five corresponding bands of Sentinel-2 and Landsat-8 OLI Images. Although the spectral consistency of the data was interpreted by cross-calibration analysis using the Pearson correlation coefficient, spatial consistency was evaluated by descriptive statistical analysis of investigated variables. In general, the highest correlation values were achieved for the images that were acquired in the spring season for almost all investigated variables. In the spring season, among the investigated variables, the Red band (B4), NDVI and EVI have the largest correlation coefficients of 0.94, 0.92 and 0.91, respectively. Regarding the spatial consistency, the mean and standard deviation values of all variables were consistent for all seasons except for the mean value of the NDVI for the fall season. As a result, if there is no atmospheric effect or data retrieval/acquisition error, either Landsat-8 or Sentinel-2 can be used as a combination or to provide the continuity data in longos monitoring applications. This study contributes to longos forest monitoring science in terms of remote sensing data analysis.
Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples often contain DNA from other sources, such as the host or the environment. The usual approach is sequencing specific hypervariable regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. Here, we aim to assess long-amplicon PCR-based approaches for assigning taxonomy at the genus and species level. We use Nanopore sequencing with two different markers: full-length 16S rRNA (~1,500 bp) and the whole rrn operon (16S rRNA–ITS–23S rRNA; 4,500 bp).
Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius , two mock communities (HM-783D, Bei Resources; D6306, ZymoBIOMICS™) and two pools of low-biomass samples (dog skin from either the chin or dorsal back), using the MinION™ sequencer 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using the WIMP workflow on EPI2ME or Minimap2 software with rrn database.
Results: The full-length 16S rRNA and the rrn operon were used to retrieve the microbiota composition at the genus and species level from the bacterial isolate, mock communities and complex skin samples. For the Staphylococcus pseudintermedius isolate, when using EPI2ME, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the rrn operon marker, and in ~68% of the cases with the 16S rRNA gene. In both skin microbiota samples, we detected many species with an environmental origin. In chin, we found different Pseudomonas species in high abundance, whereas in dorsal skin there were more taxa with lower abundances.
Conclusions: Both full-length 16S rRNA and the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, using the rrn operon would be the best choice.
The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.
A strictly anaerobic bacterial strain (SH021T) was isolated from a methanogenic reactor. Cells were Gram-stain-positive, motile, straight or slightly curved rods. The optimum temperature for growth was 35 °C, and the optimum pH was 6.1-7.7. The strain was asaccharolytic and utilized amino acids as growth substrates. The strain produced acetate and propionate from l-alanine and l-serine, and propionate and butyrate from l-threonine. Branched-chain amino acids (l-isoleucine, l-leucine and l-valine) were utilized weakly, and isovalerate or isobutyrate was produced. Strain SH021T utilized pyruvate and lactate, and converted them to acetate and propionate. The genomic DNA G+C content was 38.2 mol%. Compounds related to iso-C15 : 0 were detected as major components in the cellular fatty acids analysis. The diagnostic diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. On the basis of 16S rRNA gene sequences, the most closely related known species were Clostridium propionicum, Clostridium neopropionicum and Clostridium lactatifermentans in cluster XIVb of the class Clostridia. Based on the phylogenetic and phenotypic data, Anaerotignum aminivorans gen. nov., sp. nov. is proposed to accommodate strain SH021T (=JCM 31556T=DSM 103575T). For the three related species of the genus Clostridium, Anaerotignum propionicum comb. nov. (type strain DSM 1682T=JCM 1430T=ATCC 25522T=CCUG 9280T=NCIMB 10656T=VPI 5303T), Anaerotignum neopropionicum comb. nov. (type strain X4T=DSM 3847T=KCTC 15564T) and Anaerotignum lactatifermentans comb. nov. (type strain G17T=DSM 14214T=LMG 20954T) are proposed with emended descriptions of these species.
A Gram-stain-negative strain, A60T, isolated from a water well sample in Portugal, was characterized phenotypically, genotypically and phylogenetically. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A60T belonged to the genus Citrobacter, and recN gene phylogeny revealed one strongly supported clade encompassing strain A60T and 13 other strains from public databases, distinct from currently recognized species of the genus Citrobacter. Furthermore, multilocus sequence analysis (MLSA) based on concatenated partial fusA, leuS, pyrG and rpoB sequences confirmed the classification obtained with the recN sequence. In silico genomic comparisons, including average nucleotide identity (ANI) and the genome-to-genome distance calculator (GGDC), showed 94.6 % and 58.4 % identity to the closest relative Citrobacter freundii ATCC 8090T, respectively. The ability to metabolize different compounds further discriminated strain A60T from other species of the genus Citrobacter. The G+C content of strain A60T is 52.0 %. The results obtained support the description of a novel species within the genus Citrobacter, for which the name Citrobacter portucalensis sp. nov. is proposed, with the type strain A60T (=DSM 104542T=CECT 9236T).
Background: Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing (WGS) or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1×10⁴ bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex.
Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3,497 bidirectional high-quality (2D) reads with an average overall study length of 6,000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of mis-assigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5,500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome from MinION libraries generated using version 6 kit and chemistry yielded 899-3,497 2D reads with lengths averaging 5,700 bp with up to 98% assignment accuracy at the species-level. The observed community proportions for “equal” and “rare” synthetic libraries were close to the known proportions, deviating from 0.1 – 10% across all tests. For a 20-species mock community with staggered contributions, a sequencing run detected all but 3 species (each included at <0.05% of DNA in the total mixture); 91% of reads were assigned to the correct species, 93% of reads were assigned to the correct genus, and >99% of reads were assigned to the correct family.
Conclusions: At the current level of output and sequence quality (just under 4×10³ 2D reads for a synthetic metagenome), MinION sequencing followed by Kraken or One Codex analysis has the potential to provide rapid and accurate metagenomic analysis where the consortium is comprised of a limited number of taxa. Important considerations noted in this study included: high sensitivity of the MinION platform to the quality of input DNA, high variability of sequencing results across libraries and flow cells, and relatively small numbers of 2D reads per analysis limit. Together, these limited detection of very rare components of the microbial consortia, and would likely limit the utility of MinION for the sequencing of high-complexity metagenomic communities where thousands of taxa are expected. Furthermore, the limitations of the currently available data analysis tools suggest there is considerable room for improvement in the analytical approaches for the characterization of microbial communities using long reads. Nevertheless, the fact that the accurate taxonomic assignment of high quality reads generated by MinION is approaching 99.5% and, in most cases, the inferred community structure mirrors the known proportions of a synthetic mixture, warrants further exploration of practical application to environmental metagenomics as the platform continues to develop and improve. With further improvement in sequence throughput and error rate reduction, this platform shows great promise for precise real-time analysis of the composition and structure of more complex microbial communities.
Riverine floodplains are ecologically and economically valuable ecosystems that are heavily threatened by anthropogenic stressors. Microbial communities in floodplain soils mediate critical biogeochemical processes, yet we understand little about the relationship between these communities and variation in hydrologic connectivity related to land management or topography. Here, we present metagenomic evidence that differences among microbial communities in three floodplain soils correspond to a long-term gradient of hydrologic connectivity. Specifically, all strictly anaerobic taxa and metabolic pathways were positively associated with increased hydrologic connectivity and flooding frequency. In contrast, most aerobic taxa and all strictly aerobic pathways were negatively related to hydrologic connectivity and flooding frequency. Furthermore, the genetic potential to metabolize organic compounds tended to decrease as hydrologic connectivity increased, which may reflect either the observed concomitant decline of soil organic matter or the parallel increase in both anaerobic taxa and pathways. A decline in soil N, accompanied by an increased genetic potential for oligotrophic N acquisition subsystems, suggests that soil nutrients also shape microbial communities in these soils. We conclude that differences among floodplain soil microbial communities can be conceptualized along a gradient of hydrologic connectivity. Additionally, we show that these differences are likely due to connectivity-related variation in flooding frequency, soil organic matter, and soil N. Our findings are particularly relevant to the restoration and management of microbially mediated biogeochemical processes in riverine floodplain wetlands.
The selection of microbes by enrichment on plant biomass has been proposed as an efficient way to develop new strategies for lignocellulose saccharification. Here, we report an in-depth analysis of soil-derived microbial consortia that were trained to degrade once-used wheat straw (WS1-M), switchgrass (SG-M) and corn stover (CS-M) under aerobic and mesophilic conditions. Molecular fingerprintings, bacterial 16S ribosomal RNA (rRNA) gene amplicon sequencing and metagenomic analyses showed that the three microbial consortia were taxonomically distinct. Based on the taxonomic affiliation of protein-encoding sequences, members of the Bacteroidetes (e.g. Chryseobacterium, Weeksella, Flavobacterium and Sphingobacterium) were preferentially selected on WS1-M, whereas SG-M and CS-M favoured members of the Proteobacteria (e.g. Caulobacter, Brevundimonas, Stenotrophomonas and Xanthomonas). The highest degradation rates of lignin (~59 %) were observed with SG-M, whereas CS-M showed a high consumption of cellulose and hemicellulose. Analyses of the carbohydrate-active enzymes in the three microbial consortia showed the dominance of glycosyl hydrolases (e.g. of families GH3, GH43, GH13, GH10, GH29, GH28, GH16, GH4 and GH92). In addition, proteins of families AA6, AA10 and AA2 were detected. Analysis of secreted protein fractions (metasecretome) for each selected microbial consortium mainly showed the presence of enzymes able to degrade arabinan, arabinoxylan, xylan, β-glucan, galactomannan and rhamnogalacturonan. Notably, these metasecretomes contain enzymes that enable us to produce oligosaccharides directly from wheat straw, sugarcane bagasse and willow. Thus, the underlying microbial consortia constitute valuable resources for the production of enzyme cocktails for the efficient saccharification of plant biomass.
Strategic enrichment of microcosms derived from wood foragers can facilitate the discovery of key microbes that produce enzymes for the bioconversion of plant fiber (i.e., lignocellulose) into valuable chemicals and energy. In this study, lignocellulose-degrading microorganisms from the digestive systems of Canadian beaver (Castor canadensis) and North American moose (Alces americanus) were enriched under methanogenic conditions for over 3 years using various wood-derived substrates, including (i) cellulose (C), (ii) cellulose + lignosulphonate (CL), (iii) cellulose + tannic acid (CT), and (iv) poplar hydrolysate (PH). Substantial improvement in the conversion of amended organic substrates into biogas was observed in both beaver dropping and moose rumen enrichment cultures over the enrichment phases (up to 0.36–0.68 ml biogas/mg COD added), except for enrichments amended with tannic acid where conversion was approximately 0.15 ml biogas/mg COD added. Multiplex-pyrosequencing of 16S rRNA genes revealed systematic shifts in the population of Firmicutes, Bacteroidetes, Chlorobi, Spirochaetes, Chloroflexi, and Elusimicrobia in response to the enrichment. These shifts were predominantly substrate driven, not inoculum driven, as revealed by both UPGMA clustering pattern and OTU distribution. Additionally, the relative abundance of multiple OTUs from poorly defined taxonomic lineages increased from less than 1% to 25–50% in microcosms amended with lignocellulosic substrates, including OTUs from classes SJA-28, Endomicrobia, orders Bacteroidales, OPB54, and family Lachnospiraceae. This study provides the first direct comparison of shifts in microbial communities that occurred in different environmental samples in response to multiple relevant lignocellulosic carbon sources, and demonstrates the potential of enrichment to increase the abundance of key lignocellulolytic microorganisms and encoded activities.
Both positive and negative interactions among bacteria take place in the environment. We hypothesize that the complexity of the substrate affects the way bacteria interact with greater cooperation in the presence of recalcitrant substrate. We isolated lignocellulolytic bacteria from salt marsh detritus and compared the growth, metabolic activity and enzyme production of pure cultures to those of three-species mixed cultures in lignocellulose and glucose media. Synergistic growth was common in lignocellulose medium containing carboxyl methyl cellulose, xylan and lignin but absent in glucose medium. Bacterial synergism promoted metabolic activity in synergistic mixed cultures but not the maximal growth rate (μ). Bacterial synergism also promoted the production of β-1,4-glucosidase but not the production of cellobiohydrolase or β-1,4-xylosidase. Our results suggest that the chemical complexity of the substrate affects the way bacteria interact. While a complex substrate such as lignocellulose promotes positive interactions and synergistic growth, a labile substrate such as glucose promotes negative interactions and competition. Synergistic interactions among indigenous bacteria are suggested to be important in promoting lignocellulose degradation in the environment. © 2016, The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.
Anaerobic degradation of lignin-derived aromatics is an important metabolism for carbon and nutrient cycles in soil environments. Although there are some studies on degradation of lignin-derived aromatics by nitrate- and sulfate-reducing bacteria, knowledge on their degradation under methanogenic conditions are quite limited. In this study, methanogenic microbial communities were enriched from rice paddy field soil with lignin-derived methoxylated monoaromatics (vanillate and syringate) and their degradation intermediates (protocatechuate, catechol, and gallate) as the sole carbon and energy sources. Archaeal community analysis disclosed that both aceticlastic (Methanosarcina sp.) and hydrogenotrophic (Methanoculleus sp. and Methanocella sp.) methanogens dominated in all of the enrichments. Bacterial community analysis revealed the dominance of acetogenic bacteria (Sporomusa spp.) only in the enrichments on the methoxylated aromatics, suggesting that Sporomusa spp. initially convert vanillate and syringate into protocatechuate and gallate, respectively, with acetogenesis via O-demethylation. As the putative ring-cleavage microbes, bacteria within the phylum Firmicutes were dominantly detected from all of the enrichments, while the dominant phylotypes were not identical between enrichments on vanillate/protocatechuate/catechol (family Peptococcaceae bacteria) and on syringate/gallate (family Ruminococcaceae bacteria). This study demonstrates the importance of cooperation among acetogens, ring-cleaving fermenters/syntrophs and aceticlastic/hydrogenotrophic methanogens for degradation of lignin-derived aromatics under methanogenic conditions.
Second generation sequencing has revolutionized genomic studies. However, most genomes contain repeated DNA elements that are longer than the read lengths achievable with typical sequencers, so the genomic order of several generated contigs cannot be easily resolved. A new generation of sequencers offering substantially longer reads is emerging, notably the Pacific Biosciences (PacBio) RS II system and the MinION system, released in early 2014 by Oxford Nanopore Technologies through an early access program. The latter has highly advantageous portability and sequences samples by measuring changes in ionic current when single-stranded DNA molecules are translocated through nanopores. We show that the MinION system produces long reads with high mapability that can be used for scaffolding bacterial genomes, despite currently producing substantially higher error rates than PacBio reads. With further development we anticipate that MinION will be useful not only for assembling genomes, but also for rapid detection of organisms, potentially in the field.
Long-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments.
The assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting close to 30% of errors. Here, we presented a hybrid approach developed to take advantage of data generated using MinION® device. We sequenced a well-known bacterium, Acinetobacter baylyi ADP1 and applied our method to obtain a highly contiguous (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads.
We described NaS tool, a hybrid approach allowing the sequencing of microbial genomes using the MinION® device. Our method, based ideally on 20x and 50x of NaS and Illumina reads respectively, provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, currently with a high error rate, it is already useful in the generation of high-quality genome assemblies.
Alkaline pretreatment has the potential to enhance the anaerobic digestion of lignocellulosic biomass to biogas. However, the elevated pH of the substrate may require alkalitolerant microbial communities for an effective digestion. Three mixed anaerobic lignocellulolytic cultures were enriched from sediments from two soda lakes with wheat straw as substrate under alkaline (pH 9) mesophilic (37°C) and thermophilic (55°C) conditions. The gas production of the three cultures ceased after 4 to 5 weeks, and the produced gas was composed of carbon dioxide and methane. The main liquid intermediates were acetate and propionate. The physiological behavior of the cultures was stable even after several transfers. The enrichment process was also followed by molecular fingerprinting (terminal restriction fragment length polymorphism) of the bacterial 16S rRNA gene and of the mcrA/mrtA functional gene for methanogens. The main shift in the microbial community composition occurred between the sediment samples and the first enrichment, whereas the structure was stable in the following transfers. The bacterial communities mainly consisted of Sphingobacteriales, Clostridiales and Spirochaeta, but differed at genus level. Methanothermobacter and Methanosarcina genera and the order Methanomicrobiales were predominant methanogenes in the obtained cultures. Additionally, single cellulolytic microorganisms were isolated from enrichment cultures and identified as members of the alkaliphilic or alkalitolerant genera. The results show that anaerobic alkaline habitats harbor diverse microbial communities, which can degrade lignocellulose effectively and are therefore a potential resource for improving anaerobic digestion.
© 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
Pelosinus species can reduce metals such as Fe(III), U(VI), and Cr(VI) and have been isolated from diverse geographical regions. Five
draft genome sequences have been published. We report the complete genome sequence for Pelosinus sp. strain UFO1 using only PacBio DNA sequence data and without manual finishing.
Chitin is the second most produced biopolymer on Earth after cellulose. Chitin degrading enzymes are promising but untapped sources for developing novel industrial biocatalysts. Hidden amongst uncultivated micro-organisms, new bacterial enzymes can be discovered and exploited by metagenomic approaches through extensive cloning and screening. Enrichment is also a well-known strategy, as it allows selection of organisms adapted to feed on a specific compound. In this study, we investigated how the soil bacterial community responded to chitin enrichment in a microcosm experiment. An integrative metagenomic approach coupling phylochips and high throughput shotgun pyrosequencing was established in order to assess the taxonomical and functional changes in the soil bacterial community. Results indicate that chitin enrichment leads to an increase of Actinobacteria, γ-proteobacteria and β-proteobacteria suggesting specific selection of chitin degrading bacteria belonging to these classes. Part of enriched bacterial genera were not yet reported to be involved in chitin degradation, like the members from the Micrococcineae sub-order (Actinobacteria). An increase of the observed bacterial diversity was noticed, with detection of specific genera only in chitin treated conditions. The relative proportion of metagenomic sequences related to chitin degradation was significantly increased, even if it represents only a tiny fraction of the sequence diversity found in a soil metagenome.
Enrichment of microbial consortia provides an approach to simulate and investigate microbial communities in natural environments. In this study, a cellulolytic microbial consortium SQD-1.1 was enriched from mangrove soil of Qinglan port (Hainan, China) by 27 times continuous subcultivation under anaerobic static conditions. The consortium could completely degrade 0.2 % (w/v) filter paper within 3 days and utilized it as the sole carbon source. PCR-denaturing gradient gel electrophoresis analysis revealed a stable microbial community structure in the incubation process of 10 days and in the procedure of subcultivation. Twenty-four operational taxonomic units belonging to seven phyla were obtained from the full-length 16S rRNA gene library. Five clones, closest related to the genera Alkaliflexus, Clostridium, Alistipes, Spirochaeta, and Trichococcus, were the predominant ones. Among them, M117, phylogeneticly showing high similarity (16S rRNA gene identity, 95.3 %) with the cellulolytic anaerobic bacterium Clostridium straminisolvens CSK1(T), was the potential key cellulolytic bacterium. Using the plate cultivation method, 12 strains, including one potential new species and four potential new species of new genera, were isolated. The strain P2, corresponding to the most frequently detected clone (M05) in the 16S rRNA gene library, showed both CMCase and xylanase activity and may be another important cellulolytic bacterium. The findings of cellulase activity in cell pellet and cohesion and dockerin domains in metagenome data further suggested the potential of utilization of cellulosomes by the consortium to degrade cellulose. Consortium SQD-1.1 provides a candidate for investigating the mechanism of cellulose degradation under anoxic conditions in natural environments.
Industrial bagasse collection sites at sugar mills are an important resource for biomass-based industries and represent a unique ecological niche in lignocellulose degradation. In this study, microbial community structures at regions with varying microenvironmental conditions contained within a bagasse collection site were explored using tagged 16S rRNA gene pyrosequencing. Overall, remarkable differences in microbial community structures were found in aerobic surface and oxygen-limited interior regions of the pile. A variety of Alphaproteobacteria and Gammaproteobacteria represented the majority of bacteria in the aerobic upper-pile regions with the predominance of acetic acid bacteria towards the outer surface. Diverse Proteobacteria, Bacteroidetes, and Acidobacteria represented the predominant phyla at the exterior soil-contact pile base with an increasing abundance of anaerobic Spirochaetes with the increasing depth, where it shared similar community structures to that in the open-field soil from decomposed bagasse. Using complementary shotgun pyrosequencing, a variety of genes encoding various glycosyl hydrolases targeting cellulose and hemicellulose degradation were identified in the oxygen-limited interior pile base. Most were relevant to orders Clostridiales, Bacteroidales, Sphingobacteriales, and Cytophagales, suggesting their role in lignocellulose degradation in this region, as evidenced by the decrease in cellulose and respective increase in lignin fractions of the biomass. Partial carbon flux in the anoxic region was metabolized through mixed methanogenesis pathways as suggested by the annotated functional genes in methane synthesis. This study gives insights into native microbial community structures and functions in this unique lignocellulose degrading environment and provides the basis for controlling microbial processes important for utilization of bagasse in bio-industries.
Decomposition of lignocelluloses by cooperative microbial actions is an essential process of carbon cycling in nature and provides a basis for biomass conversion to fuels and chemicals in biorefineries. In this study, structurally stable symbiotic aero-tolerant lignocellulose-degrading microbial consortia were obtained from biodiversified microflora present in industrial sugarcane bagasse pile (BGC-1), cow rumen fluid (CRC-1), and pulp mill activated sludge (ASC-1) by successive subcultivation on rice straw under facultative anoxic conditions. Tagged 16S rRNA gene pyrosequencing revealed that all isolated consortia originated from highly diverse environmental microflora shared similar composite phylum profiles comprising mainly Firmicutes, reflecting convergent adaptation of microcosm structures, however, with substantial differences at refined genus level. BGC-1 comprising cellulolytic Clostridium and Acetanaerobacterium in stable coexistence with ligninolytic Ureibacillus showed the highest capability on degradation of agricultural residues and industrial pulp waste with CMCase, xylanase, and β-glucanase activities in the supernatant. Shotgun pyrosequencing of the BGC-1 metagenome indicated a markedly high relative abundance of genes encoding for glycosyl hydrolases, particularly for lignocellulytic enzymes in 26 families. The enzyme system comprised a unique composition of main-chain degrading and side-chain processing hydrolases, dominated by GH2, 3, 5, 9, 10, and 43, reflecting adaptation of enzyme profiles to the specific substrate. Gene mapping showed metabolic potential of BGC-1 for conversion of biomass sugars to various fermentation products of industrial importance. The symbiotic consortium is a promising simplified model for study of multispecies mechanisms on consolidated bioprocessing and a platform for discovering efficient synergistic enzyme systems for biotechnological application.
Many microorganisms contain cellulases that are important for plant cell wall degradation and overall soil ecosystem functioning. Presently, we have extensive biochemical knowledge of cellulases but little is known about the phylogenetic distribution of these enzymes. To address this, we analyzed the distribution of 21,985 genes encoding proteins related to cellulose utilization in 5,123 sequenced bacterial genomes. First, we identified the distribution of glycoside hydrolases involved in cellulose utilization and synthesis at different taxonomic levels, from the phylum to the strain. Cellulose degradation/utilization capabilities appeared in nearly all major groups and resulted in strains displaying various enzyme gene combinations. Potential cellulose degraders, having both cellulases and β-glucosidases, constituted 24% of all genomes whereas potential opportunistic strains, having β-glucosidases only, accounted for 56%. Finally, 20% of the bacteria have no relevant enzymes and do not rely on cellulose utilization. The latter group was primarily connected to specific bacterial life-styles like autotrophy and parasitism. Cellulose degraders, as well as opportunists, have multiple enzymes with similar functions. However, the potential degraders systematically harbor about twice more β-glucosidases than their potential opportunistic relatives. Although scattered, the functional types' distribution, in bacterial lineages, is not random but follow a mostly Brownian Motion evolution model. Degraders form clusters of relatives at the species level whereas opportunists are clustered at the genus level. This information can form a mechanistic basis for how to link changes in microbial community composition to soil ecosystem processes.
A novel anaerobic, rod-shaped, non-motile bacterium, designated strain ES005 T , was isolated from tidal flat sediments near the rhizosphere of Phragmites australis at Eulsukdo Island, Republic of Korea. A polyphasic approach revealed that cells of the strain were Gram-stain-positive, catalase- and oxidase-negative, non-spore-forming rods. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain ES005 T belonged to the family Eubacteriaceae , class Clostridia and showed the highest sequence similarity to Alkalibacter mobili s (97.52 %) and followed by Alkalibacter saccharofermentans Z-79820 T (96.72%). The OrthoANI value between strain ES005 T and A. mobilis was 69.67 %. Strain ES005 T grew optimally at 33–37 °C, at pH 6.0–7.0 and in the presence of 1–2 % (w/v) NaCl. Growth in 12.5 % CO atmosphere was observed. Acetate and formate were end products of fructose fermentation and growth on CO. The major cellular fatty acids of strain ES005 T were C 14 : 0 (39.1 %) and C 16 : 0 (26.6 %). The major polar lipids were diphoshatidylgycerol, phosphatidylglycerol and three unidentified phospholipids. The DNA G+C content of strain ES005 T was 46.9 mol%. Based on the phenotypic, phylogenetic, genomic and chemotaxonomic features of the isloate, strain ES005 T represents a novel species, for which the name Alkalibacter rhizosphaerae sp. nov. is proposed. The type strain is ES005 T (=KCTC 25246 T =JCM 34530 T )
The development of advanced biofuels from waste organic matter, such as lignocellulosic biomass, is critical for global sustainable waste management and to delay climate change by reducing greenhouse gas emissions via partial replacement of fossil fuels. However, the inherent recalcitrance of lignocellulosic biomass due to the presence of inhibitory components, mainly lignin, limits the hydrolysis of its carbohydrate content, representing a key hurdle augmenting biofuel production. Therefore, pretreatment of lignocellulosic biomass is crucial to promote its fragmentation, increase its surface area and solubility, and lower the cellulose
crystallinity and lignin content for sustainable biorefinery. Conventional pretreatment processes have several drawbacks, including high operational costs, corrosion of equipment, and generation of toxic effluents and by-products. To offset the negative
impacts of these limitations on biofuel production, here, we have discussed and critically compared various eco-friendly approaches for the efficient conversion of biomass to ensure high yields of biofuels as a commercial solution. Moreover, a range of microbes and enzymes have been highlighted that effectively utilize lignocellulosic biomass to obtain energy and convert its complex polymeric structure into a
biodegradable one, facilitating its subsequent valorization. Furthermore, the importance of multi-omics approaches was discussed to gain functional insights into the lignocellulolytic microbial communities and their interspecies symbiosis during the
hydrolysis of organic biomass. Finally, the limitations of previous studies, challenges, industrial perspectives, and future outlooks for the development of economical, energy-saving, and eco-friendly strategies toward the sustainable valorization of lignocellulosic biomass were summarized.
A novel anaerobic chemoorganotrophic, facultatively alkaliphilic bacterium (strain M17 DMB T ) was isolated from a coastal lake (Golubitsckoe, Taman Peninsula, Russia). Cells were motile rods, 1.6–2.1 µm long and 0.45 µm in diameter. The temperature range for growth was 14–42 °C, with an optimum at 30 °C. The pH range for growth was pH 5.5–10.0, with an optimum at pH 8.0–8.5. Growth of strain M17 DMB T was observed at NaCl concentrations of 1–12 % (w/v) with optimum growth at 1.5–2.0 %. Strain M17 MB T utilized glucose, fructose, sucrose, ribose, mannose, raffinose, arabinose, dextrin, yeast extract, peptone, carbon monoxide, vanillic acid and 3,4-dimethoxybenzoic acid. The end products from glucose fermentation were acetate and ethanol. The DNA G+C content of strain M17 DMB T was 39.1 mol%. The closest phylogenetic relative of strain M17 DMB T was Alkalibacter saccharofermentans with 97.8 % 16S rRNA gene sequence similarity. The OrthoANI value between M17 DMB T and A. saccharofermentans was 70.4 %. Based on the phenotypic, genotypic and phylogenetic characteristics of the isolate, strain M17 DMB T is considered to represent a novel species of the genus Alkalibacter for which the name Alkalibacter mobilis sp. nov. is proposed. The type strain of Alkalibacter mobilis is M17 DMB T (=KCTC 15920 T =VKM B-3408 T ).
This paper aims to contribute to the understanding of bacterial community patterns of the lakes of İğneada Floodplain Forest by metabarcoding approach. Within this scope, surface water samples were collected from three lakes located in the area namely Mert Lake, Hamam Lake, and Saka Lake, and the bacterial diversity was assessed by a high throughput sequencing of the 16S rRNA gene. Chao1 richness and Shannon diversity were higher in Saka Lake indicated a more diverse bacterial community. Proteobacteria was by far the most abundant phyla in all lakes. Although Bacteroidetes and Actinobacteria also dominated the community, their abundances differed in each lake. While the family Burkholderiaceae represented 25% of the bacterial community in Saka Lake, the abundances were 9% and 4% in Hamam Lake and Mert Lake, respectively. This study is one of the first investigations specifically focused on the bacterial communities in three lakes of İğneada Floodplain by next-generation sequencing platform and gave a prescreening of the bacterial diversity. Further studies are required to determine the biotechnological potential of this unique habitat.
River floodplains are spatially diverse ecosystems that respond quickly to flow variations and disturbance. However, it remains unclear how flow alteration and hydrological disturbance impacts the structure and biodiversity of complex microbial communities in these ecosystems. Here, we examined the spatial and seasonal dynamics of microbial communities in aquatic (benthic) and terrestrial habitats of three hydrologically contrasting (natural flow, residual flow, hydropeaking flow) floodplain systems. Microbial communities (alpha and beta diversity) differed more among floodplain habitats than between riverine floodplains. Microbial communities in all systems displayed congruent seasonal effects. In the residual and hydropeaking systems, an experimental flood was released from a reservoir to mimic a natural high flow event causing hydromorphological disturbance. The experimental flood caused a temporary shift in microbial communities by releasing microbes from the reservoir as well as redistributing communities among floodplain habitats. The flood-mediated shift in community structures had only a transient impact as pelagic bacteria did not persist within floodplain habitats over time after the flood. More frequent pulse disturbances might lead to an alternate structure of bacterial communities in floodplains over time.
Biogas production from different waste resources still has limitations due to its complex structure and slowly biodegradable nature. To improve methane yield and anaerobic digestion performance, various substrate pre-treatment methods have been suggested. This paper reviews the latest trends, progress, and research achievements about pretreatment technologies to improve anaerobic digestion efficiency. The pretreatment techniques T are divided into four main groups which are physical, chemical, biological, and combined. The effect of inhibitor formation during the pretreatment process is discussed. The energy performance, economics, and environmental impact of these pretreatment technologies are revealed. This study concludes with future trends and emphasizes the necessity of pretreatment methods.
Heterogeneity and rigidity of lignocellulose causing resistance to its deconstruction have provided technical and economic challenges in the current biomass conversion processes. Lignin has been considered as a crucial recalcitrance component in biomass utilization. An in-depth understanding of lignin properties and their influences on biomass conversion can provide clues to improve biomass utilization. Also, utilization of lignin can significantly increase the economic viability of biorefinery. Recent lignin-targeting pretreatments have aimed not only to overcome recalcitrance for biomass conversion but also to selectively fractionate lignin for lignin valorization. Numerous studies have been conducted in biomass characteristics and conversion technologies, and the role of lignin is critical for lignin valorization and biomass pretreatment development. This review provides a comprehensive review of lignin-related biomass characteristics, the impact of lignin on the biological conversion of biomass, and recent lignin-targeting pretreatment strategies. The desired lignin properties in biorefinery and future pretreatment directions are also proposed.
The presence of poorly biodegradable components in lignocellulosic biomass limits the methane recovery in anaerobic digesters. The main reason to go for aerobic pretreatment before anaerobic digestion (AD) is to enable enzymatic cleavage of the aromatic rings in lignin by oxygen since it cannot be efficiently degraded under anaerobic conditions. In this study, the advantage of highly-cellulolytic white-rot fungi Trametes versicolor was taken by aerobic pretreatment prior to anaerobic co-digestion of cow manure and selected cereal crop materials (i.e. wheat, rye, barley, triticale) harvested at different stages. Fungal pretreatment improved the methane yield by 10%-18% and cellulose degradation up to 80%. Furthermore, higher volatile fatty acid (VFA) speciation was found in the anaerobic digesters upon fungal pretreatment. 16S rRNA gene amplicon sequencing revealed a more diverse microbial community in the fungal-pretreated anaerobic digesters. Generally, typically-detected bacterial species dominated the digesters; except that Synergistetes was only enriched in the fungal-pretreated di-gesters. Although Methanosarcianease was the predominant methanogenic archaea, a more diverse methanogenic population was identified in the fungal-pretreated digesters in which Methanobacteriaceaa and Methanomibrobiaceae also took role during biomethanation. Comparatively more unique microbiome of biogas reactors upon fungal pretreatment synergistically affected VFA production, cellulose degradation and eventually methane yield in an affirmative way. Considering the functional importance of bacterial and methanogenic archaeal populations, elevated knowledge of the microbial structures is essential for minimizing process failures and for creating strategies for process optimization of lignocellulose based-AD.
The rapid depletion of natural resources and the environmental concerns associated with the use of fossil fuels as the main source of global energy is leading to an increased interest in alternative and renewable energy sources. Particular interest has been given to the lignocellulosic biomass as the most abundant source of organic matter with a potential of being utilized for energy recovery. Different approaches have been applied to convert the lignocellulosic biomass to energy products including anaerobic digestion (AD), fermentation, combustion, pyrolysis, and gasification. The AD process has been proven as an effective technology for converting organic material into energy in the form of methane-rich biogas. However, the complex structure of the lignocellulosic biomass comprised of cellulose, hemicelluloses, and lignin hinders the ability of microorganisms in an AD process to degrade and convert these compounds to biogas. Therefore, a pretreatment step is essential to improve the degradability of the lignocellulosic biomass to achieve higher biogas rate and yield. A system that uses pretreatment and AD is known as advanced AD. Several pretreatment methods have been studied over the past few years including physical, thermal, chemical and biological pretreatment. This paper reviews the enzymatic pretreatment as one of the biological pretreatment methods which has received less attention in the literature than the other pretreatment methods. This paper includes a review of lignocellulosic biomass composition, AD process, challenges in degrading lignocellulosic materials, the current status of research to improve the biogas rate and yield from the AD of lignocellulosic biomass via enzymatic pretreatment, and the future trend in research for the reduction of enzymatic pretreatment cost.
Microbial marker-gene sequence data can be used to generate comprehensive taxonomic profiles of the microorganisms present in a given community and for other community diversity analyses. The process of going from raw gene sequences to taxonomic profiles or diversity measures involves a series of data transformations performed by numerous computational tools. This includes tools for sequence quality checking, denoising, taxonomic classification, alignment, and phylogenetic tree building. In this chapter, we demonstrate how the Quantitative Insights Into Microbial Ecology version 2 (QIIME2) software suite can simplify 16S rRNA marker-gene analysis. We walk through an example data set extracted from the guts of bumblebees in order to show how QIIME2 can transform raw sequences into taxonomic bar plots, phylogenetic trees, principal co-ordinates analyses, and other visualizations of microbial diversity.
In recent years, lignin valorization is commercially an important and advanced sustainable process for lignocellulosic biomass-based industries, primarily through the depolymerization path. The conversion of the lignin moieties into biofuels and other high value-added products are still challenging to the researchers due to the heterogeneity and complex structure of lignin-containing biomass. Besides, the involvement of different microorganisms that carries varying metabolic and enzymatic complex systems towards degradation and conversion of the lignin moieties also discussed. These microorganisms are frequently short of the traits which are obligatory for the industrial application to achieve maximum yields and productivity. This review mainly focuses on the current progress and developments in the pretreatment routes for enhancing lignin degradation and also assesses the liquid and gaseous biofuel production by fermentation, gasification and hybrid technologies along with the biorefinery schemes which involves the synthesis of high value-added chemicals, biochar and other valuable products.
The present work summarizes different sources of biomass used as raw material for the production of biogas, focusing mainly on the use of plants that do not compete with the food supply. Biogas obtained from edible plants entails a developed technology and good yield of methane production; however, its use may not be sustainable. Biomass from agricultural waste is a cheap option, but in general, with lower methane yields than those obtained from edible plants. On the other hand, the use of algae or aquatic plants promises to be an efficient and sustainable option with high yields of methane produced, but it necessary to overcome the existing technological barriers. Moreover, these last raw materials have the additional advantage that they can be obtained from wastewater treatment and, therefore, they could be applied to the concept of biorefinery. An estimation of methane yield per hectare per year of the some types of biomass and operational conditions employed is presented as well. In addition, different strategies to improve the yield of biogas, such as physical, chemical, and biological pretreatments, are presented. Other alternatives for enhanced the biogas production such as bioaugmentation and biohythane are showed and finally perspectives are mentioned.
Three different bioaugmentation cultures enriched from natural and engineered cellulolytic environments (cow and goat rumen, a biogas reactor digesting sorghum biomass) were compared for their enhancement potential on the anaerobic digestion of wheat straw. Methane yields were determined in batch tests using the Automatic Methane Potential Test System operated for 30 days under mesophilic conditions. All cultures had positive effects on substrate degradation, and higher methane yields were observed in the bioaugmented reactors compared to control reactors set up with standard inoculum. However, the level of enhancement differed according to the type of the enrichment culture. Methane yield in batch reactors augmented with 2% cow rumen derived enrichment culture was increased by only 6%. In contrast, reactors amended with 2% goat rumen derived enrichment culture or with the bioaugmentation culture obtained from the biogas reactor digesting sorghum biomass produced 27% and 20% more methane, respectively. The highest methane yield was recorded in reactors amended with 6% goat rumen derived enrichment culture, which represented an increase by 36%. The microbial communities were quite similar at the end of the batch tests independently of the bioaugmentation sources, indicating that the introduced microbial communities of the enrichment cultures did not dominate the reactors.
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Currently, global demand for energy has grown and the search for new ecological energy sources is one of the mostly significant issues we face. The digestion of alternative sources of carbon in anoxic environment produces gas of high calorific value, which is a promising source of alternative energy. Thus, this work aimed to evaluate the biogas production of waste originated from a slaughterhouse industry of pigs and poultry, and from the dairy industry, and to characterize the physicochemical properties and microbiological composition of the biogas-producing biomass. Residues were collected and physicochemical and microbiological parameters were evaluated in four different stages of biogas production. At the end of 42 days, approximately 26 L of methane and 12 L of other gases were produced. The high amount of biogas/methane observed was related to the families Porphyromonadaceae, Tissierellaceae, and Methanobacteriaceae. Although less than 6% of the total reads lack classification at any taxonomic level, our analysis showed that about 50% of the sequences did not present a homologue sequence at the genus level in public databases. Knowledge about changes in the microbial composition and their dominance can provide tools for manipulation, isolation, and inoculation of the microorganisms inside the bioreactors to maximize methane production.
This study aimed to improve biomethane production from lignocellulosic biomass by assessing the impact of bioaugmentation with Clostridium thermocellum on the performance of anaerobic digesters at different inoculation ratios. The outputs of the digestion experiments revealed that bioaugmentation strategies with C. thermocellum increased the methane yield up to 39%. The sequencing analysis indicated that the indigenous microbial community was modified by the bioaugmentation. During the process of bioaugmentation, in the digester that was inoculated at the ratio of 20% (v:v), an increase in the abundance of Ruminococcaceae family led to a decrease in the Bacteroidaceae and Synergistaceae families. Furthermore, the metabolic products of the bioaugmented strains greatly influenced the diversity of the archaeal community and an increase in the abundance of Methanomicrobiales was observed.
This review commemorates the 40th anniversary of DNA sequencing, a period in which we have already witnessed multiple technological revolutions and a growth in scale from a few kilobases to the first human genome, and now to millions of human and a myriad of other genomes. DNA sequencing has been extensively and creatively repurposed, including as a 'counter' for a vast range of molecular phenomena. We predict that in the long view of history, the impact of DNA sequencing will be on a par with that of the microscope.
The aim of this study was to determine the potential of bioaugmentation with cellulolytic rumen microbiota to enhance the anaerobic digestion of lignocellulosic feedstock. An anaerobic cellulolytic culture was enriched from sheep rumen fluid using wheat straw as substrate under mesophilic conditions. To investigate the effects of bioaugmentation on methane production from straw, the enrichment culture was added to batch reactors in proportions of 2% (Set-1) and 4% (Set-2) of the microbial cell number of the standard inoculum slurry. The methane production in the bioaugmented reactors was higher than in the control reactors. After 30 days of batch incubation, the average methane yield was 154 mLN CH4 gVS⁻¹ in the control reactors. Addition of 2% enrichment culture did not enhance methane production, whereas in Set-2 the methane yield was increased by 27%. The bacterial communities were examined by 454 amplicon sequencing of 16S rRNA genes, while terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of mcrA genes was applied to analyze the methanogenic communities. The results highlighted that relative abundances of Ruminococcaceae and Lachnospiraceae increased during the enrichment. However, Cloacamonaceae, which were abundant in the standard inoculum, dominated the bacterial communities of all batch reactors. T-RFLP profiles revealed that Methanobacteriales were predominant in the rumen fluid, whereas the enrichment culture was dominated by Methanosarcinales. In the batch rectors, the most abundant methanogens were affiliated to Methanobacteriales and Methanomicrobiales. Our results suggest that bioaugmentation with sheep rumen enrichment cultures can enhance the performance of digesters treating lignocellulosic feedstock.
In this work, aspen and wheat straw substrates were pretreated using aqueous dilute acid (DA) and chemimechanical (CM) pretreatment, and the impact of these pretreatments on biomass lignin was investigated. Here DA pretreatment refers to the acidic treatment of biomass powders and CM pretreatment refers to the acidic treatments of chips or stalks followed by disc refining. The resulting substrates were observed using advanced microscopy techniques to determine changes in lignin distribution throughout the fiber cell wall due to the acid treatments. Following acid treatment, lignin within the cell wall was divided into spherical or elongated bundles. The surface of DA and CM pretreated substrates were shown to differ, with CM pretreated aspen exhibited a more cellulose-rich surface. X-ray Photoelectron Spectroscopy data indicated reduced surface lignin on CM pretreated aspen and increased surface lignin on CM pretreated wheat straw. Fourier transform infrared spectroscopy (FT-IR) spectra illustrated changes in bands related to hemicellulose lignin, and surface hydroxyl content suggesting possible differences in lignin chemistry. It was hypothesized that differences in lignin distribution based on biomass type and pretreatment method were due to interactions between the chemical treatment and the mechanical size reductions steps. Particle size prior to chemical treatment could impact the movement of lignin onto sample surface, and chemical treatment prior to size reduction could impact the fracture plane during the downsizing process. It is expected that both these effects could impact lignin distribution within the sample and on the sample surface.
The effects of pH and moisture content on high-solids sludge digestion were investigated using a mesophilic batch digester fed with sludge cake. The experiments were carried out by changing the initial moisture contents from 90 to 96% and the initial pH from 5.0 to 10.0. A simple model developed from the Gompertz equation was applied to estimate the methane production rate and the lag-phase time under various conditions, based on the cumulative methane production curves. The relative methanogenic activity decreased with the decrease of moisture content and dropped from 100 to 53% when the moisture content decreased from 96 to 90%. The rate of the methane production of the high-solids digestion at moisture contents of 90 to 96% functioned over a range of 6.6–7.8 with an optimum of pH 6.8, whereas the process may fail if the pH is lower than 6.1 or higher than 8.3. Moreover, a minimum lag-phase time for methane production was also found at around pH 6.8. In addition, a modified Haldane equation was suitable to represent the effect of pH on methanogenic activity at various moisture contents. The maximum specific methane production rate, Km, and the saturation rate constants for hydrogen ion, KH, and hydroxyl ion, KOH, were 14.5-7.5 ml CH . g−1 dry weight . d−1, 5.0 × 10−1–8.0 × 10−5 [M] and 2.0 × 10−9–8.0 × 10−9 [M], respectively.