Efficient Degradation of Lignocellulosic Plant Biomass,without Pretreatment,by the Thermophilic Anaerobe “Anaerocellum thermophilum” DSM 6725

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.Utilization of lignocellulosic biomass derived from renewable plant material to produce ethanol and other fuels is viewed as a major alternative to petroleum-based energy sources (19). The efficient conversion of plant biomass to fermentable sugars remains a formidable challenge, however, due to the recalcitrance of the insoluble starting materials (13, 21, 36). Thermal and chemical pretreatments must be used to solubilize and release the sugars, but such processes are costly and not very efficient (17, 28). Most pretreatments utilize acids, alkali, or organic solvents (39). Moreover, the plant feedstocks vary considerably in their compositions. The main components of plant biomass and the sources of the fermentable sugars, cellulose and hemicellulose, are combined with lignin, which can occupy 20% (wt/wt) or more of the plant cell wall. The development of technologies to efficiently degrade plant biomass therefore faces considerable obstacles. The discovery or engineering of new microorganisms with the ability to convert the components of lignocellulosic biomass into sugars is therefore of high priority.Not many microorganisms are able to degrade pure crystalline cellulose, and the cellulose in plant biomass has a high order of crystallinity and is even less accessible to microbial or enzymatic attack (1, 12-14). Aerobic cellulolytic microorganisms usually secrete (hemi)cellulolytic enzymes containing carbohydrate-binding modules that serve to bind the catalytic domains to insoluble substrates. On the other hand, some anaerobic bacteria and fungi produce a large extracellular multienzyme complex called the cellulosome. This binds to and efficiently degrades cellulose and other polysaccharides, although it has a limited distribution in nature (3, 7). The rate at which microorganisms degrade cellulose increases dramatically with temperature (20), but the most thermophilic cellulosome-producing bacterium that has been characterized, Clostridium thermocellum, grows optimally near only 60°C (3, 9). A few anaerobic thermophiles are known that are able to grow on crystalline cellulose even though they lack cellulosomes, and in those cases the highest optimum growth temperature is 75°C (4, 32). Biomass conversion by thermophilic anaerobic microorganisms has many potential advantages over fermentation at lower temperatures. In particular, the organisms tend to have high rates of growth and metabolism, and the processes are less prone to contamination (30).The gram-positive bacterium “Anaerocellum thermophilum” strain Z-1320 is among the most thermophilic of the cellulolytic anaerobes (32). It grows optimally at 75°C at neutral pH and utilizes both simple and complex polysaccharides, although it does not grow on xylose or pectin (32). The end products of fermentation are lactate, ethanol, acetate, CO₂, and hydrogen. Although A. thermophilum Z-1320 grows very rapidly on crystalline cellulose (4), surprisingly, it has been studied very little since its discovery (32). We report here on the physiology of a very closely related strain, A. thermophilum DSM 6725, the genome of which was recently sequenced (16). The ability of A. thermophilum DSM 6725 to grow on different types of defined and complex substrates was investigated with a focus on switchgrass and poplar. These high-lignin plants have been selected as models for biomass-to-biofuel conversion by the BioEnergy Science Center (funded by the U.S. Department of Energy; http://bioenergycenter.org/). We show that A. thermophilum DSM 6725 is able to grow efficiently on both types of plant substrate without a chemical pretreatment step.     

Cloning and expression of Clostridium thermocellum F7 cellulase genes in Escherichia coli and Bacillus subtilis cells

The Clostridium thermocellum total DNA Sau 3A fragments' library was constructed on the basis of shuttle vector pMK4 for the Escherichia coli - Bacillus subtilis. 14 clones with endoglucanase activity and one with beta-glucosidase activity were selected in E. coli cells. Recombinant plasmids pCE were characterized by structural instability of various degree in B. subtilis cells. The results of the physical mapping, analysis of gene products in E. coli mini-cells as well as the DNA-DNA blot hybridization have led to conclusion on cloning of 7 individual genes for endoglucanases. Up to 3 polypeptides of various molecular weight corresponding to the products of cel gene were revealed in E. coli mini-cells containing the recombinant plasmids. The hybridization analysis demonstrated considerable homology of the majority of cel genes.     

The vegetative state as a manifestation of a stable pathological state

The results of comprehensive monitoring of the state of 25 patients in the vegetative state (prolonged coma) before and after compensating for the factors of secondary brain damage were analyzed and followed up for no less than six months. The primary results showed that the best recovery of consciousness and cognitive functions was observed if, according to the positron emission tomography data, a diffuse decrease in the glucose metabolism rate (GMR_glu), significantly exceeding the extent of the zones of morphological/anatomical lesions, was present in the brain before treatment, while the minimal improvement was observed if the GMR_glu was sufficiently intact. These and other paradoxical results can be explained if the vegetative state is regarded as a stable pathological state of the brain, which offers new approaches to the treatment of this group of patients.     

Effect of applied and internal hormones on vitrification and apical necrosis of different plants cultured in vitro

Development of vitrification and apical necrosis was followed in Camellia sinensis, Gerbera jamesonii, Malus domestica and hybrid Populus tremula x P. alba shoots cultured in vitro on Murashige & Skoog (MS) medium with different concentrations of growth regulators. High humidity in the culture vessels and excess of BA in the medium were found to be the major factors influencing vitrification. Lack of exogenous cytokinin in the medium during successive subcultures induced apical necrosis in poor-rooting species (Malus domestica, Camellia sinensis). The level of internal phytohormones (ABA, IAA, IPA, 2iP, Z, ZR) was determined in the apple shoots by means of ELISA. The content of internal cytokinins in the vitrified apple shoots was several times greater than in normal ones, which supports the hypothesis that excess of cytokinins, inducing rapid divisions of cells in meristems in the atmosphere with high humidity, is responsible for vitrification. Apical necrosis of the plantlets that appeared after cultivation on cytokinin-free medium is the result of deficiency in endogenous hormones in apple shoots and this being confirmed by analysis of endogenous hormones in apple shoots.Abbreviations BA benzyladenine - BHT butylated hydroxy-toluene - ABA abscisic acid - IAA indole-3-acetic acid - ELISA enzyme-linked immunosorbent assay - IPA isopentenyladenosine - 2iP isopentenyladenine - NAA naphthyl-3-acetic acid - TBS trishydroxymethylaminomethane buffered saline - TLC thin layer chromatography - Z zeatin - ZR zeatin riboside