Project description:Phylogenetic, microbiological and comparative genomic analysis was used to examine the diversity among members of the genus Caldicellulosiruptor with an eye towards the capacity of these extremely thermophilic bacteria for degrading the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii (formerly Anaerocellum thermophilum), C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA phylogeny and cross-species DNA-DNA hybridization to a whole genome C. saccharolyticus oligonucleotide microarray. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid pre-treated switchgrass, only C. saccharolyticus, C. besci, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman No. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that species capable of crystalline cellulose hydrolysis also had diverse secretome fingerprints. The two-dimensional secretome of C. saccharolyticus revealed a prominent S-layer protein that appears to be also indicative of highly cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interaction. These growth physiology results were also linked to glycoside hydrolase and carbohydrate-binding module inventories for the seven bacteria, deduced from draft genome sequence information. These preliminary inventories indicated that the absence of a single glycoside hydrolase family and carbohydrate binding motif family appear to be responsible for some Caldicellulosiruptor species’ diminished cellulolytic capabilities. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and is well suited for biomass deconstruction applications.

Project description:Phylogenetic, microbiological and comparative genomic analysis was used to examine the diversity among members of the genus Caldicellulosiruptor with an eye towards the capacity of these extremely thermophilic bacteria for degrading the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii (formerly Anaerocellum thermophilum), C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA phylogeny and cross-species DNA-DNA hybridization to a whole genome C. saccharolyticus oligonucleotide microarray. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid pre-treated switchgrass, only C. saccharolyticus, C. besci, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman No. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that species capable of crystalline cellulose hydrolysis also had diverse secretome fingerprints. The two-dimensional secretome of C. saccharolyticus revealed a prominent S-layer protein that appears to be also indicative of highly cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interaction. These growth physiology results were also linked to glycoside hydrolase and carbohydrate-binding module inventories for the seven bacteria, deduced from draft genome sequence information. These preliminary inventories indicated that the absence of a single glycoside hydrolase family and carbohydrate binding motif family appear to be responsible for some Caldicellulosiruptor species’ diminished cellulolytic capabilities. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and is well suited for biomass deconstruction applications. Six dye-flip experiments were conducted using C. saccharolyticus genomic DNA as the reference in each dye-flip, and one of six different Caldicellulosiruptor spp. as a tester in each dye-flip

Project description:BackgroundConsolidated bioprocessing (CBP) of lignocellulosic biomass to ethanol using thermophilic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic enzymes. Most studies on the thermophilic CBP concentrate on co-cultivation of the thermophilic cellulolytic bacterium Clostridium thermocellum with non-cellulolytic thermophilic anaerobes at temperatures of 55°C-60°C.ResultsWe have specifically screened for cellulolytic bacteria growing at temperatures >70°C to enable direct conversion of lignocellulosic materials into ethanol. Seven new strains of extremely thermophilic anaerobic cellulolytic bacteria of the genus Caldicellulosiruptor and eight new strains of extremely thermophilic xylanolytic/saccharolytic bacteria of the genus Thermoanaerobacter isolated from environmental samples exhibited fast growth at 72°C, extensive lignocellulose degradation and high yield ethanol production on cellulose and pretreated lignocellulosic biomass. Monocultures of Caldicellulosiruptor strains degraded up to 89-97% of the cellulose and hemicellulose polymers in pretreated biomass and produced up to 72 mM ethanol on cellulose without addition of exogenous enzymes. In dual co-cultures of Caldicellulosiruptor strains with Thermoanaerobacter strains the ethanol concentrations rose 2- to 8.2-fold compared to cellulolytic monocultures. A co-culture of Caldicellulosiruptor DIB 087C and Thermoanaerobacter DIB 097X was particularly effective in the conversion of cellulose to ethanol, ethanol comprising 34.8 mol% of the total organic products. In contrast, a co-culture of Caldicellulosiruptor saccharolyticus DSM 8903 and Thermoanaerobacter mathranii subsp. mathranii DSM 11426 produced only low amounts of ethanol.ConclusionsThe newly discovered Caldicellulosiruptor sp. strain DIB 004C was capable of producing unexpectedly large amounts of ethanol from lignocellulose in fermentors. The established co-cultures of new Caldicellulosiruptor strains with new Thermoanaerobacter strains underline the importance of using specific strain combinations for high ethanol yields. These co-cultures provide an efficient CBP pathway for ethanol production and represent an ideal starting point for development of a highly integrated commercial ethanol production process.

Project description:BackgroundConsolidated bioprocessing (CBP) of lignocellulosic biomass to hydrogen offers great potential for lower cost and higher efficiency compared to processes featuring dedicated cellulase production. Current studies on CBP-based hydrogen production mainly focus on using the thermophilic cellulolytic bacterium Clostridium thermocellum and the extremely thermophilic cellulolytic bacterium Caldicellulosiruptor saccharolyticus. However, no studies have demonstrated that the strains in the genus Thermoanaerobacterium could be used as the sole microorganism to accomplish both cellulose degradation and H2 generation.ResultsWe have specifically screened for moderately thermophilic cellulolytic bacteria enabling to produce hydrogen directly from conversion of lignocellulosic materials. Three new strains of thermophilic cellulolytic bacteria in the genus Thermoanaerobacterium growing at a temperature of 60°C were isolated. All of them grew well on various plant polymers including microcrystalline cellulose, filter paper, xylan, glucose, and xylose. In particular, the isolated bacterium, designated as Thermoanaerobacterium thermosaccharolyticum M18, showed high cellulolytic activity and a high yield of H2. When it was grown in 0.5% microcrystalline cellulose, approximately 82% cellulose was consumed, and the H2 yield and maximum production rate reached 10.86 mmol/g Avicel and 2.05 mmol/L/h, respectively. Natural lignocellulosic materials without any physicochemical or biological pretreatment also supported appreciable growth of strain M18, which resulted in 56.07% to 62.71% of insoluble cellulose and hemicellulose polymer degradation in corn cob, corn stalk, and wheat straw with a yield of 3.23 to 3.48 mmol H2/g substrate and an average production rate of 0.10 to 0.13 mmol H2/L/h.ConclusionsThe newly isolated strain T. thermosaccharolyticum M18 displayed effective degradation of lignocellulose and produced large amounts of hydrogen. This is the first report of a Thermoanaerobacterium species presenting cellulolytic characteristics, and this species thus represents a novel cellulolytic bacterium distinguished from all other known cellulolytic bacteria. In comparison, the extraordinary yield and specific rate of hydrogen for strain M18 obtained from lignocellulose make it more attractive in monoculture fermentation. T. thermosaccharolyticum M18 is thus a potential candidate for rapid conversion of lignocellulose to biohydrogen in a single step.

Project description:Xylanases play a crucial role in the degradation of xylan in both terrestrial and marine environments. The endoxylanase XynB from the marine bacterium Glaciecola mesophila KMM 241 is a modular enzyme comprising a long N-terminal domain (NTD) (E44 to T562) with xylan-binding ability and a catalytic domain (CD) (T563 to E912) of glycoside hydrolase family 8 (GH8). In this study, the long NTD is confirmed to contain three different functional regions, which are NTD1 (E44 to D136), NTD2 (Y137 to A193), and NTD3 (L194 to T562). NTD1, mainly composed of eight ?-strands, functions as a new type of carbohydrate-binding module (CBM), which has xylan-binding ability but no sequence similarity to any known CBM. NTD2, mainly forming two ?-helices, contains one of the ?-helices of the catalytic domain's (?/?)6 barrel and therefore is essential for the activity of XynB, although it is far away from the catalytic domain in sequence. NTD3, next to the catalytic domain in sequence, is shown to be helpful in maintaining the thermostability of XynB. Thus, XynB represents a kind of xylanase with a new domain architecture. There are four other predicted glycoside hydrolase sequences with the same domain architecture and high sequence identity (?80%) with XynB, all of which are from marine bacteria. Phylogenetic analysis shows that XynB and these homologs form a new group in GH8, representing a new class of marine bacterial xylanases. Our results shed light on xylanases, especially marine xylanases.IMPORTANCE Xylanases play a crucial role in natural xylan degradation and have been extensively used in industries such as food processing, animal feed, and kraft pulp biobleaching. Some marine bacteria have been found to secrete xylanases. Characterization of novel xylanases from marine bacteria has significance for both the clarification of xylan degradation mechanisms in the sea and the development of new enzymes for industrial application. With G. mesophila XynB as a representative, this study reveals a new group of the GH8 xylanases from marine bacteria, which have a distinct domain architecture and contain a novel carbohydrate-binding module. Thus, this study offers new knowledge on marine xylanases.

Dataset Information

Synergism of glycoside hydrolase secretomes from two thermophilic bacteria cocultivated on lignocellulose.

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Synergism of glycoside hydrolase secretomes from two thermophilic bacteria cocultivated on lignocellulose.

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