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1.  Efficient Degradation of Lignocellulosic Plant Biomass, without Pretreatment, by the Thermophilic Anaerobe “Anaerocellum thermophilum” DSM 6725▿  
Applied and Environmental Microbiology  2009;75(14):4762-4769.
Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that “Anaerocellum thermophilum” DSM 6725, an anaerobic bacterium that grows optimally at 75°C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75°C for 18 h also served as a growth substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first- and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70°C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms.
doi:10.1128/AEM.00236-09
PMCID: PMC2708433  PMID: 19465524
2.  Single-step ethanol production from lignocellulose using novel extremely thermophilic bacteria 
Background
Consolidated 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.
Results
We 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.
Conclusions
The 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.
doi:10.1186/1754-6834-6-31
PMCID: PMC3598825  PMID: 23448304
Anaerobic; Caldicellulosiruptor; Consolidated bioprocessing; Ethanol; Extremely thermophilic bacteria; High temperature; Lactate; Lignocellulose; Thermoanaerobacter
3.  Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725 
Nucleic Acids Research  2011;39(8):3240-3254.
Caldicellulosiruptor bescii DSM 6725 utilizes various polysaccharides and grows efficiently on untreated high-lignin grasses and hardwood at an optimum temperature of ∼80°C. It is a promising anaerobic bacterium for studying high-temperature biomass conversion. Its genome contains 2666 protein-coding sequences organized into 1209 operons. Expression of 2196 genes (83%) was confirmed experimentally. At least 322 genes appear to have been obtained by lateral gene transfer (LGT). Putative functions were assigned to 364 conserved/hypothetical protein (C/HP) genes. The genome contains 171 and 88 genes related to carbohydrate transport and utilization, respectively. Growth on cellulose led to the up-regulation of 32 carbohydrate-active (CAZy), 61 sugar transport, 25 transcription factor and 234 C/HP genes. Some C/HPs were overproduced on cellulose or xylan, suggesting their involvement in polysaccharide conversion. A unique feature of the genome is enrichment with genes encoding multi-modular, multi-functional CAZy proteins organized into one large cluster, the products of which are proposed to act synergistically on different components of plant cell walls and to aid the ability of C. bescii to convert plant biomass. The high duplication of CAZy domains coupled with the ability to acquire foreign genes by LGT may have allowed the bacterium to rapidly adapt to changing plant biomass-rich environments.
doi:10.1093/nar/gkq1281
PMCID: PMC3082886  PMID: 21227922
4.  Hydrogenomics of the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus▿ †  
Applied and Environmental Microbiology  2008;74(21):6720-6729.
Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO2, and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocellulose-utilizing, biofuel-producing bacterium.
doi:10.1128/AEM.00968-08
PMCID: PMC2576683  PMID: 18776029
5.  Novel monosaccharide fermentation products in Caldicellulosiruptor saccharolyticus identified using NMR spectroscopy 
Background
Caldicellulosiruptor saccharolyticus is a thermophilic, Gram-positive, non-spore forming, strictly anaerobic bacterium of interest in potential industrial applications, including the production of biofuels such as hydrogen or ethanol from lignocellulosic biomass through fermentation. High-resolution, solution-state nuclear magnetic resonance (NMR) spectroscopy is a useful method for the identification and quantification of metabolites that result from growth on different substrates. NMR allows facile resolution of isomeric (identical mass) constituents and does not destroy the sample.
Results
Profiles of metabolites produced by the thermophilic cellulose-degrading bacterium Caldicellulosiruptor saccharolyticus DSM 8903 strain following growth on different monosaccharides (D-glucose, D-mannose, L-arabinose, D-arabinose, D-xylose, L-fucose, and D-fucose) as carbon sources revealed several unexpected fermentation products, suggesting novel metabolic capacities and unexplored metabolic pathways in this organism. Both 1H and 13C nuclear magnetic resonance (NMR) spectroscopy were used to determine intracellular and extracellular metabolite profiles. One dimensional 1H NMR spectral analysis was performed by curve fitting against spectral libraries provided in the Chenomx software; 2-D homonuclear and heteronuclear NMR experiments were conducted to further reduce uncertainties due to unassigned, overlapping, or poorly-resolved peaks. In addition to expected metabolites such as acetate, lactate, glycerol, and ethanol, several novel fermentation products were identified: ethylene glycol (from growth on D-arabinose), acetoin and 2,3-butanediol (from growth on D-glucose, L-arabinose, and D-xylose), and hydroxyacetone (from growth on D-mannose, L-arabinose, and D-xylose). Production of ethylene glycol from D-arabinose was particularly notable, with around 10% of the substrate carbon converted into this uncommon fermentation product.
Conclusions
The present research shows that C. saccharolyticus, already of substantial interest due to its capability for biological ethanol and hydrogen production, has further metabolic potential for production of higher molecular weight compounds, such as acetoin and 2,3-butanediol, as well as hydroxyacetone and the uncommon fermentation product ethylene glycol. In addition, application of nuclear magnetic resonance (NMR) spectroscopy facilitates identification of novel metabolites, which is instrumental for production of desirable bioproducts from biomass through microbial fermentation.
doi:10.1186/1754-6834-6-47
PMCID: PMC3637100  PMID: 23552326
Caldicellulosiruptor saccharolyticus; Nuclear magnetic resonance; Bioproducts; Acetoin; 2,3-Butanediol; Ethylene glycol
6.  S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus 
The genus Caldicellulosiruptor contains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes of Caldicellulosiruptor species reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins in Caldicellulosiruptor saccharolyticus (Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequenced Caldicellulosiruptor species. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in the C. saccharolyticus genome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to the C. saccharolyticus S-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in other Caldicellulosiruptor genomes may also be important contributors to plant biomass utilization.
doi:10.1128/AEM.07031-11
PMCID: PMC3264102  PMID: 22138994
7.  Single-step bioconversion of lignocellulose to hydrogen using novel moderately thermophilic bacteria 
Background
Consolidated 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.
Results
We 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.
Conclusions
The 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.
doi:10.1186/1754-6834-7-82
PMCID: PMC4052809  PMID: 24920960
Lignocellulose; Thermoanaerobacterium thermosaccharolyticum; Biohydrogen; Degradation; Consolidated bioprocessing
8.  Phylogenetic, Microbiological, and Glycoside Hydrolase Diversities within the Extremely Thermophilic, Plant Biomass-Degrading Genus Caldicellulosiruptor▿  
Applied and Environmental Microbiology  2010;76(24):8084-8092.
Phylogenetic, microbiological, and comparative genomic analyses were used to examine the diversity among members of the genus Caldicellulosiruptor, with an eye toward the capacity of these extremely thermophilic bacteria to degrade the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii, C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA gene phylogeny and cross-species DNA-DNA hybridization to a whole-genome C. saccharolyticus oligonucleotide microarray, revealing that C. saccharolyticus was the most divergent within this group. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid-pretreated switchgrass, only C. saccharolyticus, C. bescii, 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 the cellulolytic species also had diverse secretome fingerprints. The C. saccharolyticus secretome contained a prominent S-layer protein that appears in the cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interactions. Growth physiology also correlated with glycoside hydrolase (GH) and carbohydrate-binding module (CBM) inventories for the seven bacteria, as deduced from draft genome sequence information. These inventories indicated that the absence of a single GH and CBM family was responsible for diminished cellulolytic capacity. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and this argues for continued efforts to isolate new members from high-temperature terrestrial biotopes.
doi:10.1128/AEM.01400-10
PMCID: PMC3008241  PMID: 20971878
9.  Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Noncellulosomal Thermophilic Deconstruction of Plant Biomass 
Journal of Bacteriology  2012;194(15):4015-4028.
Extremely thermophilic bacteria of the genus Caldicellulosiruptor utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acid-pretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse Caldicellulosiruptor species. The open Caldicellulosiruptor pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic Caldicellulosiruptor species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose.
doi:10.1128/JB.00266-12
PMCID: PMC3416521  PMID: 22636774
10.  Carbohydrate Utilization Patterns for the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus Reveal Broad Growth Substrate Preferences ▿ †  
Applied and Environmental Microbiology  2009;75(24):7718-7724.
Coutilization of hexoses and pentoses derived from lignocellulose is an attractive trait in microorganisms considered for consolidated biomass processing to biofuels. This issue was examined for the H2-producing, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus growing on individual monosaccharides (arabinose, fructose, galactose, glucose, mannose, and xylose), mixtures of these sugars, as well as on xylan and xylogluco-oligosacchrides. C. saccharolyticus grew at approximately the same rate (td, ∼95 min) and to the same final cell density (1 × 108 to 3 × 108 cells/ml) on all sugars and sugar mixtures tested. In the monosaccharide mixture, although simultaneous consumption of all monosaccharides was observed, not all were utilized to the same extent (fructose > xylose/arabinose > mannose/glucose/galactose). Transcriptome contrasts for monosaccharide growth revealed minimal changes in some cases (e.g., 32 open reading frames [ORFs] changed ≥2-fold for glucose versus galactose), while substantial changes occurred for cases involving mannose (e.g., 353 ORFs changed ≥2-fold for glucose versus mannose). Evidence for catabolite repression was not noted for either growth on multisugar mixtures or the corresponding transcriptomes. Based on the whole-genome transcriptional response analysis and comparative genomics, carbohydrate specificities for transport systems could be proposed for most of the 24 putative carbohydrate ATP-binding cassette transporters and single phosphotransferase system identified in C. saccharolyticus. Although most transporter genes responded to individual monosaccharides and polysaccharides, the genes Csac_0692 to Csac_0694 were upregulated only in the monosaccharide mixture. The results presented here affirm the broad growth substrate preferences of C. saccharolyticus on carbohydrates representative of lignocellulosic biomass and suggest that this bacterium holds promise for biofuel applications.
doi:10.1128/AEM.01959-09
PMCID: PMC2794124  PMID: 19820143
11.  Biohydrogen Production by the Thermophilic Bacterium Caldicellulosiruptor saccharolyticus: Current Status and Perspectives 
Life : Open Access Journal  2013;3(1):52-85.
Caldicellulosiruptor saccharolyticus is one of the most thermophilic cellulolytic organisms known to date. This Gram-positive anaerobic bacterium ferments a broad spectrum of mono-, di- and polysaccharides to mainly acetate, CO2 and hydrogen. With hydrogen yields approaching the theoretical limit for dark fermentation of 4 mol hydrogen per mol hexose, this organism has proven itself to be an excellent candidate for biological hydrogen production. This review provides an overview of the research on C. saccharolyticus with respect to the hydrolytic capability, sugar metabolism, hydrogen formation, mechanisms involved in hydrogen inhibition, and the regulation of the redox and carbon metabolism. Analysis of currently available fermentation data reveal decreased hydrogen yields under non-ideal cultivation conditions, which are mainly associated with the accumulation of hydrogen in the liquid phase. Thermodynamic considerations concerning the reactions involved in hydrogen formation are discussed with respect to the dissolved hydrogen concentration. Novel cultivation data demonstrate the sensitivity of C. saccharolyticus to increased hydrogen levels regarding substrate load and nitrogen limitation. In addition, special attention is given to the rhamnose metabolism, which represents an unusual type of redox balancing. Finally, several approaches are suggested to improve biohydrogen production by C. saccharolyticus.
doi:10.3390/life3010052
PMCID: PMC4187192  PMID: 25371332
Caldicellulosiruptor saccharolyticus; biohydrogen; dark fermentation; cellulolytic thermophile; thermodynamics; rhamnose metabolism; pyrophosphate; redox balance; hydrogen inhibition; regulation
12.  Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalis generates a new host for the analysis of biomass deconstruction 
Biotechnology for Biofuels  2014;7(1):132.
Background
Members of the thermophilic, anaerobic Gram-positive bacterial genus Caldicellulosiruptor grow optimally at 65 to 78°C and degrade lignocellulosic biomass without conventional pretreatment. Decomposition of complex cell wall polysaccharides is a major bottleneck in the conversion of plant biomass to biofuels and chemicals, and conventional biomass pretreatment includes exposure to high temperatures, acids, or bases as well as enzymatic digestion. Members of this genus contain a variety of glycosyl hydrolases, pectinases, and xylanases, but the contribution of these individual enzymes to biomass deconstruction is largely unknown. C. hydrothermalis is of special interest because it is the least cellulolytic of all the Caldicellulosiruptor species so far characterized, making it an ideal naïve system to study key cellulolytic enzymes from these bacteria.
Results
To develop methods for genetic manipulation of C. hydrothermalis, we selected a spontaneous deletion of pyrF, a gene in the pyrimidine biosynthetic pathway, resulting in a strain that was a uracil auxotroph resistant to 5-fluoroorotic acid (5-FOA). This strain allowed the selection of prototrophic transformants with either replicating or non-replicating plasmids containing the wild-type pyrF gene. Counter-selection of the pyrF wild-type allele on non-replicating vectors allowed the construction of chromosomal deletions. To eliminate integration of the non-replicating plasmid at the pyrF locus in the C. hydrothermalis chromosome, we used the non-homologous Clostridium thermocellum wild-type pyrF allele to complement the C. hydrothermalis pyrF deletion. The autonomously replicating shuttle vector was maintained at 25 to 115 copies per chromosome. Deletion of the ChyI restriction enzyme in C. hydrothermalis increased the transformation efficiency by an order of magnitude and demonstrated the ability to construct deletions and insertions in the genome of this new host.
Conclusions
The use of C. hydrothermalis as a host for homologous and heterologous expression of enzymes important for biomass deconstruction will enable the identification of enzymes that contribute to the special ability of these bacteria to degrade complex lignocellulosic substrates as well as facilitate the construction of strains to improve and extend their substrate utilization capabilities.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-014-0132-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13068-014-0132-8
PMCID: PMC4172971  PMID: 25254074
13.  Genome-wide analysis of acetivibrio cellulolyticus provides a blueprint of an elaborate cellulosome system 
BMC Genomics  2012;13:210.
Background
Microbial degradation of plant cell walls and its conversion to sugars and other byproducts is a key step in the carbon cycle on Earth. In order to process heterogeneous plant-derived biomass, specialized anaerobic bacteria use an elaborate multi-enzyme cellulosome complex to synergistically deconstruct cellulosic substrates. The cellulosome was first discovered in the cellulolytic thermophile, Clostridium thermocellum, and much of our knowledge of this intriguing type of protein composite is based on the cellulosome of this environmentally and biotechnologically important bacterium. The recently sequenced genome of the cellulolytic mesophile, Acetivibrio cellulolyticus, allows detailed comparison of the cellulosomes of these two select cellulosome-producing bacteria.
Results
Comprehensive analysis of the A. cellulolyticus draft genome sequence revealed a very sophisticated cellulosome system. Compared to C. thermocellum, the cellulosomal architecture of A. cellulolyticus is much more extensive, whereby the genome encodes for twice the number of cohesin- and dockerin-containing proteins. The A. cellulolyticus genome has thus evolved an inflated number of 143 dockerin-containing genes, coding for multimodular proteins with distinctive catalytic and carbohydrate-binding modules that play critical roles in biomass degradation. Additionally, 41 putative cohesin modules distributed in 16 different scaffoldin proteins were identified in the genome, representing a broader diversity and modularity than those of Clostridium thermocellum. Although many of the A. cellulolyticus scaffoldins appear in unconventional modular combinations, elements of the basic structural scaffoldins are maintained in both species. In addition, both species exhibit similarly elaborate cell-anchoring and cellulosome-related gene- regulatory elements.
Conclusions
This work portrays a particularly intricate, cell-surface cellulosome system in A. cellulolyticus and provides a blueprint for examining the specific roles of the various cellulosomal components in the degradation of complex carbohydrate substrates of the plant cell wall by the bacterium.
doi:10.1186/1471-2164-13-210
PMCID: PMC3413522  PMID: 22646801
Cellulosomics; Clostridium thermocellum; Scaffoldin; Cohesin; Dockerin
14.  Stable coexistence of two Caldicellulosiruptor species in a de novo constructed hydrogen-producing co-culture 
Background
Mixed culture enrichments have been used frequently for biohydrogen production from different feedstock. In spite of the several advantages offered by those cultures, they suffer poor H2 yield. Constructing defined co-cultures of known H2 producers may offer a better performance than mixed-population enrichments, while overcoming some of the limitations of pure cultures based on synergies among the microorganisms involved.
Results
The extreme thermophiles Caldicellulosiruptor saccharolyticus DSM 8903 and C. kristjanssonii DSM 12137 were combined in a co-culture for H2 production from glucose and xylose in a continuous-flow stirred tank reactor. The co-culture exhibited a remarkable stability over a period of 70 days under carbon-sufficient conditions, with both strains coexisting in the system at steady states of different dilution rates, as revealed by species-specific quantitative PCR assays. The two strains retained their ability to stably coexist in the reactor even when glucose was used as the sole growth-limiting substrate. Furthermore, H2 yields on glucose exceeded those of either organism alone under the same conditions, alluding to a synergistic effect of the two strains on H2 production. A maximum H2 yield of 3.7 mol (mol glucose)-1 was obtained by the co-culture at a dilution rate of 0.06 h-1; a higher yield than that reported for any mixed culture to date. A reproducible pattern of population dynamics was observed in the co-culture under both carbon and non-carbon limited conditions, with C. kristjanssonii outgrowing C. saccharolyticus during the batch start-up phase and prevailing at higher dilution rates. A basic continuous culture model assuming the ability of C. saccharolyticus to enhance the growth of C. kristjanssonii could mimic the pattern of population dynamics observed experimentally and provide clues to the nature of interaction between the two strains. As a proof, the cell-free growth supernatant of C. saccharolyticus was found able to enhance the growth of C. kristjanssonii in batch culture through shortening its lag phase and increasing its maximum biomass concentration by ca. 18%.
Conclusions
This study provides experimental evidence on the stable coexistence of two closely related organisms isolated from geographically-distant habitats under continuous operation conditions, with the production of H2 at high yields. An interspecies interaction is proposed as the reason behind the remarkable ability of the two Caldicellulosiruptor strains to coexist in the system rather than only competing for the growth-limiting substrate.
doi:10.1186/1475-2859-9-102
PMCID: PMC3022713  PMID: 21192828
15.  Complete Genome Sequences for the Anaerobic, Extremely Thermophilic Plant Biomass-Degrading Bacteria Caldicellulosiruptor hydrothermalis, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor kronotskyensis, Caldicellulosiruptor owensensis, and Caldicellulosiruptor lactoaceticus▿  
Journal of Bacteriology  2011;193(6):1483-1484.
The genus Caldicellulosiruptor contains the most thermophilic, plant biomass-degrading bacteria isolated to date. Previously, genome sequences from three cellulolytic members of this genus were reported (C. saccharolyticus, C. bescii, and C. obsidiansis). To further explore the physiological and biochemical basis for polysaccharide degradation within this genus, five additional genomes were sequenced: C. hydrothermalis, C. kristjanssonii, C. kronotskyensis, C. lactoaceticus, and C. owensensis. Taken together, the seven completed and one draft-phase Caldicellulosiruptor genomes suggest that, while central metabolism is highly conserved, significant differences in glycoside hydrolase inventories and numbers of carbohydrate transporters exist, a finding which likely relates to variability observed in plant biomass degradation capacity.
doi:10.1128/JB.01515-10
PMCID: PMC3067630  PMID: 21216991
16.  Proteome-wide systems analysis of a cellulosic biofuel-producing microbe 
We apply mass spectrometry-based ReDi proteomics to quantify the Clostridium phytofermentans proteome during fermentation of cellulosic substrates. ReDi proteomics gives accurate, low-cost quantification of an extra and intracellular microbial proteome. When combined with physiological measurements, these methods form a general systems biology strategy to evaluate the efficiency of cellulosic bioconversion and to identify enzyme targets to engineer for improving this process.C. phytofermentans expressed more than 100 carbohydrate-active enzymes, of which distinct subsets were upregulated on cellulose and hemicellulose. Numerous extracellular enzymes cleave insoluble plant polysaccharides into oligosaccharides, which are transported into the cell to be further degraded by intracellular carbohydratases. Sugars are catabolized by EMP glycolysis incorporating alternative glycolytic enzymes to maximize the ATP yield of anaerobic metabolism.During cellulosic fermentation, cells adhered to the substrate and altered metabolic processes such as upregulation of tryptophan and nicotinamide synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. These diverse metabolic changes highlight how a systems approach can identify novel ways to optimize cellulosic fermentation.
Cellulose is the world's most abundant renewable, biological energy source (Leschine, 1995). Microbial fermentation of cellulosic biomass could sustainably provide enough ethanol for 65% of US ground transportation fuel at current levels (Somerville, 2006). However, cellulose in plant biomass is packaged into a crystalline matrix, making biomass deconstruction a key roadblock to using it as a feedstock (Houghton et al, 2006). A promising strategy to overcome biomass recalcitrance is consolidated bioprocessing (Lynd et al, 2002), which uses microbes such as Clostridium phytofermentans to both secrete enzymes to depolymerize biomass and then ferment the resulting hexose and pentose sugars to a biofuel such as ethanol. The C. phytofermentans genome encodes 161 carbohydrate-active enzymes (CAZy) including 108 glycoside hydrolases spread across 39 families (Cantarel et al, 2009), highlighting the elaborate set of enzymes needed to breakdown different cellulosic polysaccharides. Faced with the complexity of metabolizing biomass, systems biology strategies are needed to comprehensively identify which cellulolytic and metabolic enzymes are used to ferment different cellulosic substrates.
This study presents a systems-level analysis of how C. phytofermentans ferments different cellulosic substrates that incorporates quantitative mass spectrometry-based proteomics of over 2500 proteins. Protein concentrations within each carbon source treatment were calculated by machine learning-supported spectral counting (Absolute Protein EXpression, APEX) (Lu et al, 2007). Protein levels on hemicellulose and cellulose relative to glucose were determined using reductive methylation (Hsu et al, 2003; Boersema et al, 2009), here called ReDi labeling, to chemically incorporate hydrogen or deuterium isotopes at lysines and N-terminal amines of tryptic peptides. We show that ReDi proteomics gives accurate, low-cost quantification of a microbial proteome and can be used to discern extracellular proteins. Further, we combine these quantitative proteomics with detailed measurements of growth, biomass consumption, fermentation product analyses, and electron microscopy. Together, these methods form a general strategy to evaluate the efficiency of cellulosic bioconversion and to identify enzyme targets to engineer for improving this process (Figure 1).
We found that fermentation of cellulosic substrates by C. phytofermentans involves secretion of numerous CAZy as well as proteins for binding of extracellular solutes, proteolysis, and motility. The most highly expressed protein in the proteome is a secreted protein that appears to compose a surface layer to support the cell and anchor cell surface proteins, including some enzymes for plant degradation. Once the secreted CAZy cleave insoluble plant polysaccharides into oligosaccharides, they are taken into the cell to be further degraded by intracellular CAZy, enabling more efficient sugar transport, conserving energy by phosphorolytic cleavage, and ensuring the sugar monomers were not available to competing microbes. Sugars are catabolized by EMP glycolysis incorporating reversible, PPi-dependent glycolytic enzymes, and pyruvate ferredoxin oxidoreductase. The genome encodes seven alcohol dehydrogenases, among which two iron-dependent enzymes are highly expressed and likely facilitate the high ethanol yields. Growth on cellulose also resulted in indirect changes such as increased tryptophan and nicotinamide synthesis and repression of fatty acid synthesis. We distilled the data into a model showing the highly expressed enzymes enabling efficient cellulosic fermentation by C. phytofermentans (Figure 7). Collectively, these data help understand how bacteria recycle plant biomass works towards enabling the use of plant biomass as a low-cost chemical feedstock.
Fermentation of plant biomass by microbes like Clostridium phytofermentans recycles carbon globally and can make biofuels from inedible feedstocks. We analyzed C. phytofermentans fermenting cellulosic substrates by integrating quantitative mass spectrometry of more than 2500 proteins with measurements of growth, enzyme activities, fermentation products, and electron microscopy. Absolute protein concentrations were estimated using Absolute Protein EXpression (APEX); relative changes between treatments were quantified with chemical stable isotope labeling by reductive dimethylation (ReDi). We identified the different combinations of carbohydratases used to degrade cellulose and hemicellulose, many of which were secreted based on quantification of supernatant proteins, as well as the repertoires of glycolytic enzymes and alcohol dehydrogenases (ADHs) enabling ethanol production at near maximal yields. Growth on cellulose also resulted in diverse changes such as increased expression of tryptophan synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. This study gives a systems-level understanding of how this microbe ferments biomass and provides a rational, empirical basis to identify engineering targets for industrial cellulosic fermentation.
doi:10.1038/msb.2010.116
PMCID: PMC3049413  PMID: 21245846
bioenergy; clostridium; proteomics
17.  Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass 
Biotechnology for Biofuels  2014;7(1):142.
Background
Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic organisms described to date, and have the ability to grow on lignocellulosic biomass without conventional pretreatment. Different species vary in their abilities to degrade cellulose, and the presence of CelA, a bifunctional glycoside hydrolase that contains a Family 48 and a Family 9 catalytic domain, correlates well with cellulolytic ability in members of this genus. For example, C. hydrothermalis, which does not contain a CelA homolog, or a GH48 Family or GH9 Family glycoside hydrolase, is the least cellulolytic of the Caldicellulosiruptor species so far described. C. bescii, which contains CelA and expresses it constitutively, is among the most cellulolytic. In fact, CelA is the most abundant extracellular protein produced in C. bescii. The enzyme contains two catalytic units, a Family 9A-CBM3c processive endoglucanase and a Family 48 exoglucanase, joined by two Family 3b carbohydrate-binding domains. Although there are two non-reducing end-specific Family 9 and three reducing end-specific Family 48 glycoside hydrolases (producing primarily glucose and cellobiose; and cellobiose and cellotriose, respectively) in C. bescii, CelA is the only protein that combines both enzymatic activities.
Results
A deletion of the celA gene resulted in a dramatic reduction in the microorganism’s ability to grow on crystalline cellulose (Avicel) and diminished growth on lignocellulosic biomass. A comparison of the overall endoglucanase and exoglucanase activities of the mutant compared with the wild-type suggests that the loss of the endoglucanase activity provided by the GH9 family domain is perhaps compensated for by other enzymes produced by the cell. In contrast, it appears that no other enzymes in the C. bescii secretome can compensate for the loss of exoglucanase activity. The change in enzymatic activity in the celA mutant resulted in a 15-fold decrease in sugar release on Avicel compared with the parent and wild-type strains.
Conclusions
The exoglucanase activity of the GH48 domain of CelA plays a major role in biomass degradation within the suite of C. bescii biomass-degrading enzymes.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-014-0142-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13068-014-0142-6
PMCID: PMC4195899  PMID: 25317205
Bioenergy; Cellulase; Thermophile
18.  Deconstruction of Lignocellulose into Soluble Sugars by Native and Designer Cellulosomes 
mBio  2012;3(6):e00508-12.
ABSTRACT
Lignocellulosic biomass, the most abundant polymer on Earth, is typically composed of three major constituents: cellulose, hemicellulose, and lignin. The crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix are three major factors that contribute to the observed recalcitrance of lignocellulose. By means of designer cellulosome technology, we can overcome the recalcitrant properties of lignocellulosic substrates and thus increase the level of native enzymatic degradation. In this context, we have integrated six dockerin-bearing cellulases and xylanases from the highly cellulolytic bacterium, Thermobifida fusca, into a chimeric scaffoldin engineered to bear a cellulose-binding module and the appropriate matching cohesin modules. The resultant hexavalent designer cellulosome represents the most elaborate artificial enzyme composite yet constructed, and the fully functional complex achieved enhanced levels (up to 1.6-fold) of degradation of untreated wheat straw compared to those of the wild-type free enzymes. The action of these designer cellulosomes on wheat straw was 33 to 42% as efficient as the natural cellulosomes of Clostridium thermocellum. In contrast, the reduction of substrate complexity by chemical or biological pretreatment of the substrate removed the advantage of the designer cellulosomes, as the free enzymes displayed higher levels of activity, indicating that enzyme proximity between these selected enzymes was less significant on pretreated substrates. Pretreatment of the substrate caused an increase in activity for all the systems, and the native cellulosome completely converted the substrate into soluble saccharides.
IMPORTANCE
Cellulosic biomass is a potential alternative resource which could satisfy future demands of transportation fuel. However, overcoming the natural lignocellulose recalcitrance remains challenging. Current research and development efforts have concentrated on the efficient cellulose-degrading strategies of cellulosome-producing anaerobic bacteria. Cellulosomes are multienzyme complexes capable of converting the plant cell wall polysaccharides into soluble sugar products en route to biofuels as an alternative to fossil fuels. Using a designer cellulosome approach, we have constructed the largest form of homogeneous artificial cellulosomes reported to date, which bear a total of six different cellulases and xylanases from the highly cellulolytic bacterium Thermobifida fusca. These designer cellulosomes were comparable in size to natural cellulosomes and displayed enhanced synergistic activities compared to their free wild-type enzyme counterparts. Future efforts should be invested to improve these processes to approach or surpass the efficiency of natural cellulosomes for cost-effective production of biofuels.
doi:10.1128/mBio.00508-12
PMCID: PMC3520109  PMID: 23232718
19.  The Genome Sequences of Cellulomonas fimi and “Cellvibrio gilvus” Reveal the Cellulolytic Strategies of Two Facultative Anaerobes, Transfer of “Cellvibrio gilvus” to the Genus Cellulomonas, and Proposal of Cellulomonas gilvus sp. nov 
PLoS ONE  2013;8(1):e53954.
Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484T. For comparative purposes, we also sequenced the genome of the aerobic cellulolytic “Cellvibrio gilvus” ATCC 13127T. An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that “Cellvibrio gilvus” belongs to the genus Cellulomonas. We thus propose to assign “Cellvibrio gilvus” to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482T showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.
doi:10.1371/journal.pone.0053954
PMCID: PMC3544764  PMID: 23342046
20.  Enhanced whole genome sequence and annotation of Clostridium stercorarium DSM8532T using RNA-seq transcriptomics and high-throughput proteomics 
BMC Genomics  2014;15(1):567.
Background
Growing interest in cellulolytic clostridia with potential for consolidated biofuels production is mitigated by low conversion of raw substrates to desired end products. Strategies to improve conversion are likely to benefit from emerging techniques to define molecular systems biology of these organisms. Clostridium stercorarium DSM8532T is an anaerobic thermophile with demonstrated high ethanol production on cellulose and hemicellulose. Although several lignocellulolytic enzymes in this organism have been well-characterized, details concerning carbohydrate transporters and central metabolism have not been described. Therefore, the goal of this study is to define an improved whole genome sequence (WGS) for this organism using in-depth molecular profiling by RNA-seq transcriptomics and tandem mass spectrometry-based proteomics.
Results
A paired-end Roche/454 WGS assembly was closed through application of an in silico algorithm designed to resolve repetitive sequence regions, resulting in a circular replicon with one gap and a region of 2 kilobases with 10 ambiguous bases. RNA-seq transcriptomics resulted in nearly complete coverage of the genome, identifying errors in homopolymer length attributable to 454 sequencing. Peptide sequences resulting from high-throughput tandem mass spectrometry of trypsin-digested protein extracts were mapped to 1,755 annotated proteins (68% of all protein-coding regions). Proteogenomic analysis confirmed the quality of annotation and improvement pipelines, identifying a missing gene and an alternative reading frame. Peptide coverage of genes hypothetically involved in substrate hydrolysis, transport and utilization confirmed multiple pathways for glycolysis, pyruvate conversion and recycling of intermediates. No sequences homologous to transaldolase, a central enzyme in the pentose phosphate pathway, were observed by any method, despite demonstrated growth of this organism on xylose and xylan hemicellulose.
Conclusions
Complementary omics techniques confirm the quality of genome sequence assembly, annotation and error-reporting. Nearly complete genome coverage by RNA-seq likely indicates background DNA in RNA extracts, however these preps resulted in WGS enhancement and transcriptome profiling in a single Illumina run. No detection of transaldolase by any method despite xylose utilization by this organism indicates an alternative pathway for sedoheptulose-7-phosphate degradation. This report combines next-generation omics techniques to elucidate previously undefined features of substrate transport and central metabolism for this organism and its potential for consolidated biofuels production from lignocellulose.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-567) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-567
PMCID: PMC4102724  PMID: 24998381
Genome; Proteome; Transcriptome; RNA-seq; Tandem mass spectrometry; Proteogenomics; Glycolysis; Pentose phosphate pathway; Transaldolase
21.  Cellulolytic potential of thermophilic species from four fungal orders 
AMB Express  2013;3:47.
Elucidation of fungal biomass degradation is important for understanding the turnover of biological materials in nature and has important implications for industrial biomass conversion. In recent years there has been an increasing interest in elucidating the biological role of thermophilic fungi and in characterization of their industrially useful enzymes. In the present study we investigated the cellulolytic potential of 16 thermophilic fungi from the three ascomycete orders Sordariales, Eurotiales and Onygenales and from the zygomycete order Mucorales thus covering all fungal orders that include thermophiles. Thermophilic fungi are the only described eukaryotes that can grow at temperatures above 45°C. All 16 fungi were able to grow on crystalline cellulose but their secreted enzymes showed widely different cellulolytic activities, pH optima and thermostabilities. Interestingly, in contrast to previous reports, we found that some fungi such as Melanocarpus albomyces readily grew on crystalline cellulose and produced cellulases. These results indicate that there are large differences in the cellulolytic potential of different isolates of the same species. Furthermore, all the selected species were able to degrade cellulose but the differences in cellulolytic potential and thermostability of the secretome did not correlate to the taxonomic position. PCR amplification and sequencing of 22 cellulase genes from the fungi showed that the level of thermostability of the cellulose-degrading activity could not be inferred from the phylogenetic relationship of the cellulases.
doi:10.1186/2191-0855-3-47
PMCID: PMC3766086  PMID: 23958135
Thermophilic fungi; Endoglucanase; Cellobiohydrolase; Cellulolytic potential
22.  The Complete Genome Sequence of Fibrobacter succinogenes S85 Reveals a Cellulolytic and Metabolic Specialist 
PLoS ONE  2011;6(4):e18814.
Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.
doi:10.1371/journal.pone.0018814
PMCID: PMC3079729  PMID: 21526192
23.  Chimeric Lactase Capable of Spontaneous and Strong Immobilization on Cellulose and Development of a Continuous-Flow System for Lactose Hydrolysis at High Temperatures▿  
Applied and Environmental Microbiology  2010;76(24):8071-8075.
Recombinant plasmids containing fusion proteins composed of two different modules were constructed and expressed in Escherichia coli. The modules encoded the lactase LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus and the cellulase CelD, a cellulose-binding module (CBM) from Anaerocellum thermophilum. The CelD CBM provides a spontaneous and strong sorption of the fusion proteins onto a cellulose carrier. The enzymatic activities of both the free LacA protein and LacA-CelD CBM fusion proteins immobilized onto the cellulose carrier were assessed. The LacA activity of the fusion protein was dependent upon its position with respect to the CBM. The highest level of lactase activity and stability was observed when the lactase domain was localized at its N terminus. A continuous-flow column reactor of lactase immobilized on a cellulose carrier was constructed, and its activity was assessed. The lactose hydrolysis rate for a 150 mM (5%) solution at a flow rate of 1 reactor volume per min was 75%, which is a value optimal for further whey transformation into glucose/galactose syrup.
doi:10.1128/AEM.01517-10
PMCID: PMC3008238  PMID: 20935120
24.  Efficient hydrogen production from the lignocellulosic energy crop Miscanthus by the extreme thermophilic bacteria Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana 
Background
The production of hydrogen from biomass by fermentation is one of the routes that can contribute to a future sustainable hydrogen economy. Lignocellulosic biomass is an attractive feedstock because of its abundance, low production costs and high polysaccharide content.
Results
Batch cultures of Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana produced hydrogen, carbon dioxide and acetic acid as the main products from soluble saccharides in Miscanthus hydrolysate. The presence of fermentation inhibitors, such as furfural and 5-hydroxylmethyl furfural, in this lignocellulosic hydrolysate was avoided by the mild alkaline-pretreatment conditions at a low temperature of 75°C. Both microorganisms simultaneously and completely utilized all pentoses, hexoses and oligomeric saccharides up to a total concentration of 17 g l-1 in pH-controlled batch cultures. T. neapolitana showed a preference for glucose over xylose, which are the main sugars in the hydrolysate. Hydrogen yields of 2.9 to 3.4 mol H2 per mol of hexose, corresponding to 74 to 85% of the theoretical yield, were obtained in these batch fermentations. The yields were higher with cultures of C. saccharolyticus compared to T. neapolitana. In contrast, the rate of substrate consumption and hydrogen production was higher with T. neapolitana. At substrate concentrations exceeding 30 g l-1, sugar consumption was incomplete, and lower hydrogen yields of 2.0 to 2.4 mol per mol of consumed hexose were obtained.
Conclusion
Efficient hydrogen production in combination with simultaneous and complete utilization of all saccharides has been obtained during the growth of thermophilic bacteria on hydrolysate of the lignocellulosic feedstock Miscanthus. The use of thermophilic bacteria will therefore significantly contribute to the energy efficiency of a bioprocess for hydrogen production from biomass.
doi:10.1186/1754-6834-2-12
PMCID: PMC2701949  PMID: 19534765
25.  Aerobic deconstruction of cellulosic biomass by an insect-associated Streptomyces 
Scientific Reports  2013;3:1030.
Streptomyces are best known for producing antimicrobial secondary metabolites, but they are also recognized for their contributions to biomass utilization. Despite their importance to carbon cycling in terrestrial ecosystems, our understanding of the cellulolytic ability of Streptomyces is currently limited to a few soil-isolates. Here, we demonstrate the biomass-deconstructing capability of Streptomyces sp. SirexAA-E (ActE), an aerobic bacterium associated with the invasive pine-boring woodwasp Sirex noctilio. When grown on plant biomass, ActE secretes a suite of enzymes including endo- and exo-cellulases, CBM33 polysaccharide-monooxygenases, and hemicellulases. Genome-wide transcriptomic and proteomic analyses, and biochemical assays have revealed the key enzymes used to deconstruct crystalline cellulose, other pure polysaccharides, and biomass. The mixture of enzymes obtained from growth on biomass has biomass-degrading activity comparable to a cellulolytic enzyme cocktail from the fungus Trichoderma reesei, and thus provides a compelling example of high cellulolytic capacity in an aerobic bacterium.
doi:10.1038/srep01030
PMCID: PMC3538285  PMID: 23301151

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