Papers by Vladimir Zverlov
Biotechnology for biofuels, 2017
Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organi... more Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA. Although all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (i...
International journal of systematic and evolutionary microbiology, Jan 4, 2016
A novel Gram-stain positive, rod-shaped, anaerobic thermophilic bacterium, strain GGR1T, was isol... more A novel Gram-stain positive, rod-shaped, anaerobic thermophilic bacterium, strain GGR1T, was isolated from a thermophilic lab scale biogas fermenter. The novel organism was effectively degrading crystalline cellulose. It seems to play a role in remineralization of plant biomass by hydrolyzing its polysaccharides. 16S rRNA gene comparative sequence analysis demonstrated that the isolate formed a hitherto unknown subline within the family Ruminococcaceae. The closest relative of GGR1T among the validly described taxa is Clostridium thermocellum, sharing 94.3 % 16S rRNA gene sequence similarity. Strain GGR1T is catalase-negative, indole-negative, and produces acetate and ethanol as major end products during fermentative cellulose utilization. The major cellular fatty acids (>1 %) were 16:0 iso fatty acid and 16:0 fatty acid. Cells were rod shaped, and grew optimally at 60 °C and pH 7.0. The DNA G + C content is 34.9 mol%. A novel genus and species, Herbivorax saccincola gen. nov., s...
Biotechnology for Biofuels, 2016
Background: One of the most promising technologies to sustainably produce energy and to mitigate ... more Background: One of the most promising technologies to sustainably produce energy and to mitigate greenhouse gas emissions from combustion of fossil energy carriers is the anaerobic digestion and biomethanation of organic raw material and waste towards biogas by highly diverse microbial consortia. In this context, the microbial systems ecology of thermophilic industrial-scale biogas plants is poorly understood. Results: The microbial community structure of an exemplary thermophilic biogas plant was analyzed by a comprehensive approach comprising the analysis of the microbial metagenome and metatranscriptome complemented by the cultivation of hydrolytic and acido-/acetogenic Bacteria as well as methanogenic Archaea. Analysis of metagenome-derived 16S rRNA gene sequences revealed that the bacterial genera Defluviitoga (5.5 %), Halocella (3.5 %), Clostridium sensu stricto (1.9 %), Clostridium cluster III (1.5 %), and Tepidimicrobium (0.7 %) were most abundant. Among the Archaea, Methanoculleus (2.8 %) and Methanothermobacter (0.8 %) were predominant. As revealed by a metatranscriptomic 16S rRNA analysis, Defluviitoga (9.2 %), Clostridium cluster III (4.8 %), and Tepidanaerobacter (1.1 %) as well as Methanoculleus (5.7 %) mainly contributed to these sequence tags indicating their metabolic activity, whereas Hallocella (1.8 %), Tepidimicrobium (0.5 %), and Methanothermobacter (<0.1 %) were transcriptionally less active. By applying 11 different cultivation strategies, 52 taxonomically different microbial isolates representing the classes Clostridia, Bacilli, Thermotogae, Methanomicrobia and Methanobacteria were obtained. Genome analyses of isolates support the finding that, besides Clostridium thermocellum and Clostridium stercorarium, Defluviitoga tunisiensis participated in the hydrolysis of hemicellulose producing ethanol, acetate, and H 2 /CO 2. The latter three metabolites are substrates for hydrogentrophic and acetoclastic archaeal methanogenesis. Conclusions: Obtained results showed that high abundance of microorganisms as deduced from metagenome analysis does not necessarily indicate high transcriptional or metabolic activity, and vice versa. Additionally, it appeared that the microbiome of the investigated thermophilic biogas plant comprised a huge number of up to now unknown and insufficiently characterized species.
Genome announcements, Jan 23, 2016
The novel mesophilic bacterial strain Propionispora sp. 2/2-37 was isolated from an industrial-sc... more The novel mesophilic bacterial strain Propionispora sp. 2/2-37 was isolated from an industrial-scale biogas plant. Comparative 16S rRNA gene sequencing revealed that the isolate constitutes a new subcluster within the order Selenomonadales The 2/2-37 draft genome sequence was established and provides the genetic basis for application of this microorganism in degradation of biomass for bio-fuel production.
Genome announcements, Jan 21, 2016
A novel cellulolytic bacterial strain was isolated from an industrial-scale biogas plant. The 16S... more A novel cellulolytic bacterial strain was isolated from an industrial-scale biogas plant. The 16S rRNA gene sequence of the strain SD1D showed 96.4% similarity to Herbinix hemicellulosilytica T3/55(T), indicating a novel species within the genus Herbinix (family Lachnospiraceae). Here, the complete genome sequence of Herbinix luporum SD1D is reported.
Genome announcements, Jan 23, 2015
The draft genome sequence of Ruminoclostridium sp. Ne3 was reconstructed from the metagenome of a... more The draft genome sequence of Ruminoclostridium sp. Ne3 was reconstructed from the metagenome of a hydrogenogenic microbial consortium growing on xylan. The organism is likely the primary hemicellulose degrader within the consortium.
International journal of systematic and evolutionary microbiology, Jan 14, 2015
Phenotypic and phylogenetic studies were performed on new isolates of a novel gram-positive, anae... more Phenotypic and phylogenetic studies were performed on new isolates of a novel gram-positive, anaerobic, non sporulating rod-shaped bacterium isolated from a thermophilic biogas plant. The novel organisms were able to degrade crystalline cellulose. 16S rRNA gene comparative sequence analysis demonstrated that the isolates formed a hitherto unknown subline within the family Lachnospiraceae. As a representative of the whole group of isolates, strain T3/55T was further characterized. The closest relative of T3/55T among the validly described taxa is Mobilitalea sibirica, sharing 93.9% 16S rRNA gene sequence similarity. Strain T3/55T was catalase-negative, indole-negative, and produced acetate, ethanol, and propionic acid as major end products from cellulose metabolism. The major cellular fatty acids (>1%) were 16:0 dimethyl acetal, 16:0 fatty acid methyl ester and 16:0 aldehyde. The DNA G + C content was 36.6 mol%. A novel genus and species, Herbinix hemicellulosilytica gen. nov., sp...
Molecular Biology, 2012
At the C-terminus of multimodular laminarinase Lic16A Clostridium thermocellum four carbohydrate-... more At the C-terminus of multimodular laminarinase Lic16A Clostridium thermocellum four carbohydrate-binding modules (CBM), belonging to family 4, were found. The isolated CBM - CBM4_1, CBM4_2, CBM4_3, CBM4_4 and the tandem CBM4_(1-4) were obtained. None of the recombinant proteins did have the affinity to soluble beta-1,3-1,4-glucans--laminarin and lihenan--the main specific substrates of Licl6A. All modules, except CBM4_4, had the ability to bind bacterial crystalline cellulose, that was atypical for the family 4 CBMs. We found that all CBMs 4 of Licl6A had affinity for xylan, chitin, beta-glucan from yeast cell wall and Avicel, while CBM4_3 and CBM4_4 had additional affinity to chitosan. The tandem CBM4_(1-4) had the highest affinity to yeast cell wall beta-glucan, avicel and pustulan. The binding constants for these substrates were about 100 times higher than that of the individual modules, suggesting a synergy in the process of absorption to these polysaccharides. This finding helps to explain the evolutionary process of CBM multiplication.
Molecular Biology, 2013
The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicel... more The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second encoding a 749 aa multimodular endo 1,4 β glucanase CelD (85019 Da). The N terminal region of the protein includes a signal peptide and a catalytic module of glyco side hydrolase family 5 (GH5), followed by a carbohydrate binding module of family 28 (CBM28). The C terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic module and CBM28, were produced in E. coli cells and purified to homogeneity. An analysis of the catalytic properties showed CelD to be an endo 1,4 β glucanase with maximum activity on barley β glucan at pH 6.2 and 70°C. The enzyme was stable at 50°C for 30 days. Upon removal of the C terminal CBM28, the activity of GH5 was decreased on cellulose substrates, and its thermostability has dropped. Binding of CBM28 to amorphous cellulose has been almost irreversible as it could not be removed from this substrate in a range of pH of 4-11, temperatures of 0-75°C, and NaCl concentrations of 0-5 M. Only 100% formamide or 1% SDS have been able to remove the protein.
Applied Microbiology and Biotechnology, 1996
The nucleotide sequence of the xynA gene, encoding extracellular xylanase A of Thermotoga neapoli... more The nucleotide sequence of the xynA gene, encoding extracellular xylanase A of Thermotoga neapolitana, was determined. The xynA gene was 3264 base pairs (bp) long and encoded a putative polypeptide of 1055 amino acids. Three different domains were identified by sequence comparison and functional analysis of proteins with N-and/or C-terminal deletions. The core domain displayed significant homology to members of the glycosyl hydrolase family 10. N-and C-terminal domains were dispensable for enzymatic activity and seemed to be responsible for thermostability and cellulose binding, respectively. The intact gene and its truncated variants were expressed in Escherichia coli and purified for biochemical characterization. The enzyme was shown to act as an endo-1,4-/3-xylanase, but minor activities against lichenan, barley glucan, methylumbelliferyl cellobioside and p-nitrophenyl xyloside were also detected. The specific activity and pH and temperature optima for hydrolysis of oat xylan were 111.3 U • mg-', 5.5 and 102 C, respectively. The endoxylanase was stable at 90'C and retained 50% activity when incubated for 2 h at 100°C.
International Journal of Systematic and Evolutionary Microbiology
Applied Microbiology and Biotechnology
Applied Microbiology and Biotechnology
Butanol is a platform chemical that is utilized in a wide range of industrial products and is con... more Butanol is a platform chemical that is utilized in a wide range of industrial products and is considered a suitable replacement or additive to liquid fuels. So far, it is mainly produced through petrochemical routes. Alternative production routes, for example through biorefinery, are under investigation but are currently not at a market competitive level. Possible alternatives, such as acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia are not market-ready to this day either, because of their low butanol titer and the high costs of feedstocks. Here, we analyzed wheat middlings and wheat red dog, two wheat milling byproducts available in large quantities, as substrates for clostridial ABE fermentation. We could identify ten strains that exhibited good butanol yields on wheat red dog. Two of the best ABE producing strains, Clostridium beijerinckii NCIMB 8052 and Clostridium diolis DSM 15410, were used to optimize a laboratory-scale fermentation process. In addition...
Microorganisms
Members of the genera Proteiniphilum and Petrimonas were speculated to represent indicators refle... more Members of the genera Proteiniphilum and Petrimonas were speculated to represent indicators reflecting process instability within anaerobic digestion (AD) microbiomes. Therefore, Petrimonas mucosa ING2-E5AT was isolated from a biogas reactor sample and sequenced on the PacBio RSII and Illumina MiSeq sequencers. Phylogenetic classification positioned the strain ING2-E5AT in close proximity to Fermentimonas and Proteiniphilum species (family Dysgonomonadaceae). ING2-E5AT encodes a number of genes for glycosyl-hydrolyses (GH) which are organized in Polysaccharide Utilization Loci (PUL) comprising tandem susCD-like genes for a TonB-dependent outer-membrane transporter and a cell surface glycan-binding protein. Different GHs encoded in PUL are involved in pectin degradation, reflecting a pronounced specialization of the ING2-E5AT PUL systems regarding the decomposition of this polysaccharide. Genes encoding enzymes participating in amino acids fermentation were also identified. Fragment ...
Microorganisms
Genomic studies revealed the glycoside hydrolases of family 48 (GH48) as a powerful marker for th... more Genomic studies revealed the glycoside hydrolases of family 48 (GH48) as a powerful marker for the identification of truly cellulolytic bacteria. Here we report an improved method for detecting cellulolytic bacteria in lab-scale biogas fermenters by using GH48 genes as a molecular marker in DNA and RNA samples. We developed a mixture of primers for the specific amplification of a GH48 gene region in a broad range of bacteria. Additionally, we built a manually curated reference database containing GH48 gene sequences directly linked to the corresponding taxonomic information. Phylogenetic correlation analysis of GH48 to 16S rRNA gene sequences revealed that GH48 gene sequences with 94% identity belong with high confidence to the same genus. Applying this analysis, GH48 amplicon reads revealed that at mesophilic fermenter conditions, 50–99% of the OTUs appear to belong to novel taxa. In contrast, at thermophilic conditions, GH48 gene sequences from the genus Hungateiclostridium domina...
Microorganisms
Bacterial hydrolysis of polysaccharides is an important step for the production of sustainable en... more Bacterial hydrolysis of polysaccharides is an important step for the production of sustainable energy, for example during the conversion of plant biomass to methane-rich biogas. Previously, Hungateiclostridium thermocellum was identified as cellulolytic key player in thermophilic biogas microbiomes with a great frequency as an accompanying organism. The aim of this study was to physiologically characterize a recently isolated co-culture of H. thermocellum and the saccharolytic bacterium Defluviitalea raffinosedens from a laboratory-scale biogas fermenter. The characterization focused on cellulose breakdown by applying the measurement of cellulose hydrolysis, production of metabolites, and the activity of secreted enzymes. Substrate degradation and the production of volatile metabolites was considerably enhanced when both organisms acted synergistically. The metabolic properties of H. thermocellum have been studied well in the past. To predict the role of D. raffinosedens in this bac...
Systematic and Applied Microbiology
Biotechnology for Biofuels
Background: Glycoside hydrolases are important for various industrial and scientific applications... more Background: Glycoside hydrolases are important for various industrial and scientific applications. Determination of their temperature as well as pH optima and range is crucial to evaluate whether an enzyme is suitable for application in a biotechnological process. These basic characteristics of enzymes are generally determined by two separate measurements. However, these lead to a two-dimensional assessment of the pH range at one temperature (and vice versa) and do not allow prediction of the relative enzymatic performance at any pH/temperature combination of interest. In this work, we demonstrate a new method that is based on experimental data and visualizes the relationship among pH, temperature, and activity at a glance in a three-dimensional contour plot. Results: In this study, we present a method to determine the relative activity of an enzyme at 96 different combinations of pH and temperature in parallel. For this purpose, we used a gradient PCR cycler and a citrate-phosphatebased buffer system in microtiter plates. The approach was successfully tested with various substrates and diverse assays for glycoside hydrolases. Furthermore, its applicability was demonstrated for single enzymes using the endoglucanase Cel8A from Clostridium thermocellum as well as the commercially available complex enzyme mixture Celluclast ®. Thereby, we developed a fast and adaptable method to determine simultaneously both pH and temperature ranges of enzymes over a wide range of conditions, an easy transformation of the experimental data into a contour plot for visualization, and the necessary controls. With our method, the suitability of an enzyme or enzyme mixture for any chosen combination of temperature and pH can easily be assessed at a glance. Conclusions: We propose a method that offers significant advantages over commonly used methods to determine the pH and temperature ranges of enzymes. The overall relationship among pH, temperature, and activity is visualized. Our method could be applied to evaluate exactly what conditions have to be met for optimal utilization of an enzyme or enzyme mixture for both lab-scale and industrial processes. Adaptation to other enzymes, including proteases, should be possible and the method may also lead to a platform for additional applications, such as inactivation kinetics analysis.
Biotechnology for Biofuels
Background: Increasing the efficiency of enzymatic biomass degradation is crucial for a more econ... more Background: Increasing the efficiency of enzymatic biomass degradation is crucial for a more economically feasible conversion of abundantly available plant feedstock. Synergistic effects between the enzymes deployed in the hydrolysis of various hemicelluloses have been demonstrated, which can reduce process costs by lowering the amount of enzyme required for the reaction. Xyloglucan is the only major hemicellulose for which no such effects have been described yet. Results: We report the beneficial combination of two enzymes for the degradation of the hemicellulose xyloglucan. The addition of β-galactosidase Bga2B from Clostridium stercorarium to an in vitro hydrolysis reaction of a model xyloglucan substrate increased the enzymatic efficiency of endoglucanase Cel9D from Clostridium thermocellum to up to 22-fold. Furthermore, the total amount of enzyme required for high hydrolysis yields was lowered by nearly 80%. Increased yields were also observed when using a natural complex substrate-tamarind kernel powder. Conclusion: The findings of this study may improve the valorization of feedstocks containing high-xyloglucan amounts. The combination of the endoglucanase Cel9D and the β-galactosidase Bga2B can be used to efficiently produce the heptasaccharide XXXG. The exploitation of one specific oligosaccharide may open up possibilities for the use as a prebiotic or platform chemical in additional reactions.
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Papers by Vladimir Zverlov