Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

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Microbial cellulose utilization: fundamentals and biotechnology.

The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.

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Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Industrial applications of microbial lipases

Xylanases are hydrolytic enzymes which randomly cleave the β 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics. Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent. Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases. Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed. The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented.

Xylanases, xylanase families and extremophilic xylanases

In 1910, Ehrlich introduced the compound 3-amino-4hydroxyphenylarsenic(i) (salvarsan, arsphenamine, or Ehrlich 606) as a remedy for syphilis, a disease caused by the spirochaete bacterium Treponema pallidum. His methodical search for a specific curative for an identified disease can be regarded as the introduction of targeted chemotherapy. Despite its long history and importance, the actual structure of salvarsan is still debated. 2] Ehrlich synthesized the compound by the reaction of 3-nitro-4-hydroxyphenylarsonic acid with dithionite. This simultaneously reduced the NO2 group to NH2 and As V to As to give a material of formula 3-H2N-4-HOC6H3As (Scheme 1). The product was isolated as the hydrated hydrochloride salt of formula 3-H2N4-HOC6H3As·HCl·H2O. This synthesis was not always reproducible, and inevitably gave rise to sulfur-containing impurities, which may have accounted for the variable toxicity of different batches of salvarsan. A two-step process that involved initial reduction of the NO2 group by sodium dithionite followed by reduction of As with hypophosphorous acid was subsequently shown by Christiansen to lead to sulfur-free material (Scheme 1). By analogy with azo compounds, Ehrlich assigned structure 1 to the free base of salvarsan. Although As=As bonds are now known to be found only in sterically crowded molecules, the As=As form is repeatedly cited, with textbooks and reviews still giving structure 1. 2] Various suggestions for the true structure of salvarsan have been proposed, including large polycyclic molecules and polymers, but lack strong supporting data. We now report the first definitive evidence for the composition of salvarsan based on electrospray ionization mass spectrometric data. Salvarsan was synthesized by using a modification of the two-step synthesis first described by Christiansen. The compound was isolated as the hydrochloride, giving consistent yields of a pale-yellow material that was analyzed as the monohydrate, corresponding exactly to Ehrlich s original reports. We also isolated the mixed H2PO2 /H2PO3 salt form (P NMR spectroscopic data) by direct precipitation from the reaction mixture. H and C NMR spectroscopic data were recorded for salvarsan, but were not particularly useful for structural assignment; ESI MS proved much more practical for this purpose. A typical spectrum is shown in Figure 1. Although there are many peaks, they can be readily assigned. Three clear series can be identified (Table 1). The first of these is the major one and consists of [(RAs)n+H] + peaks (R= 3-H2N-4HOC6H3) for n= 3–8. Peaks corresponding to n= 3 and n= 5 are clearly dominant in this series. A lesser series comprises ions generated by loss of R from (RAs)n, that is, [Rn 1Asn] +

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The Composition of Ehrlich's Salvarsan: Resolution of a Century‐Old Debate

A new obligately anaerobic, extremely thermophilic, cellulolytic bacterium is described. The strain designated Tp8T 6331 is differentiated from thermophilic cellulolytic clostridia on the basis of physiological characteristics and phylogenetic position within the Bacillus/Clostridium subphylum of the Gram-positive bacteria. Strain Tp8T 6331 is assigned to a new genus Caldicellulosiruptor, as Caldicellulosiruptor saccharolyticus gen., nov., sp. nov.

Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium

16S-rDNA analysis of Spirochaeta thermophila: Its phylogenetic position and implications for the systematics of the order Spirochaetales

A new species of extremely thermophilic, glycolytic anaerobic bacterium, Fervidobacterium nodosum isolated from a New Zealand hot spring, is described. Fervidobacterium nodosum strains were Gram-negative, motile, non-sporulating obligately anaerobic rods that existed singly, in pairs or in chains. Electron micrographs of thin sections revealed a two-layered cell wall structure. The outer layer of the cell wall produced “spheroids”, which was a typical feature of this organism. The optimum temperature for growth was 65 to 70° C, the maximum 80° C and the minimum greater than 40° C. Growth occurred between a pH of 6.0 and 8.0 with the optimum being 7.0 to 7.5. The doubling time of Fervidobacterium nodosum at optimal temperature and pH was 105 minutes. The DNA base composition was 33.7% guanine plus cytosine as determined by thermal denaturation. A wide range of carbohydrates including glucose, sucrose, starch and lactose could be utilized by the organism. Lactate, acetate, hydrogen, and carbon dioxide were the major end products of glucose fermentation with lesser amounts of ethanol being formed. Growth was inhibited by tetracycline, penicillin and chloramphenicol indicating that the organism was a eubacterium.

Fervidobacterium nodosum gen. nov. and spec. nov., a new chemoorganotrophic, caldoactive, anaerobic bacterium

The loss of activity due to proteolysis of purified L-asparaginase and beta-galactosidase from different sources correlates with the thermal instability of the enzymes. A similar correlation is found when populations of soluble proteins from micro-organisms grown at different temperatures are compared for proteolytic susceptibility and thermal stability. It is proposed that there is a general correlation between the thermostability of proteins and their resistance to proteolysis.

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Hugh W. Morgan

Papers

The Composition of Ehrlich's Salvarsan: Resolution of a Century‐Old Debate

Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium

16S-rDNA analysis of Spirochaeta thermophila: Its phylogenetic position and implications for the systematics of the order Spirochaetales

Fervidobacterium nodosum gen. nov. and spec. nov., a new chemoorganotrophic, caldoactive, anaerobic bacterium

A correlation between protein thermostability and resistance to proteolysis.