Today, biomass covers about 10% of the world's primary energy demand. Against a backdrop of rising crude oil prices, depletion of resources, political instability in producing countries and environmental challenges, besides efficiency and intelligent use, only biomass has the potential to replace the supply of an energy hungry civilisation. Plant biomass is an abundant and renewable source of energy-rich carbohydrates which can be efficiently converted by microbes into biofuels, of which, only bioethanol is produced on an industrial scale today. Biomethane is produced on a large scale, but is not yet utilised for transportation. Biobutanol is on the agenda of several companies and may be used in the near future as a supplement for gasoline, diesel and kerosene, as well as contributing to the partially biological production of butyl-t-butylether, BTBE as does bioethanol today with ETBE. Biohydrogen, biomethanol and microbially made biodiesel still require further development. This paper reviews microbially made biofuels which have potential to replace our present day fuels, either alone, by blending, or by chemical conversion. It also summarises the history of biofuels and provides insight into the actual production in various countries, reviewing their policies and adaptivity to the energy challenges of foreseeable future.

Clostridial acetone-butanol fermentation from renewable carbohydrates used to be the largest biotechnological process second only to yeast ethanol fermentation and the largest process ever run under sterile conditions. With the rising prices for mineral oil, it has now the economical and technological potential to replace petrochemistry for the production of fuels from renewable resources. Various methods for using non-food biomass such as cellulose and hemicellulose in agricultural products and wastes have been developed at laboratory scale. To our knowledge, the AB plants in Russia were the only full-scale industrial plants which used hydrolyzates of lignocellosic waste for butanol fermentation. These plants were further developed into the 1980s, and the process was finally run in a continual mode different from plants in Western countries. A biorefinery concept for the use of all by-products has been elaborated and was partially put into practice. The experience gained in the Soviet Union forms a promising basis for the development of modern large-scale processes to replace a considerable fraction of the current chemical production of fuel for our future needs on a sustainable basis.

Clostridium thermocellum produces the most efficient enzyme-complex for the degradation of polysaccharides in biomass, the large extracellular cellulosome. The draft complete genomic sequence of Clostridium thermocellum was screened for open reading frames (ORF) containing cellulosomal dockerin sequences. Seventy-one putative cellulosomal genes were detected. One third of these ORFs may be involved in cellulose hydrolysis. Most of the others showed homology to hemicellulases, pectinases, chitinases, glycosidases or esterases potentially involved in the unwrapping of cellulose fibers. To identify the predominant catalytic components, cellulosomes were purified and the components were separated by an adapted two-dimensional gel electrophoresis technique. The apparent major spots were identified by MALDI-TOF/TOF. Ten of the components were previously known: the structural protein CipA, the endo-glucanases Cel8A, Cel5G, Cel9N, the cellobiohydrolases Cbh9A, Cel9K, Cel48S, the xylanases Xyn10C, Xyn10Z, and the chitinase Chi18A. In addition, three hitherto unknown major components were detected, Cel9R, Xyn10D and Xgh74A. These major components in the cellulosomal particles most probably constitute the essential enzymes for crystalline cellulose hydrolysis.

In contrast to previous findings, we demonstrate that the dissimilatory (bi)sulfite reductase genes (dsrAB) of Desulfobacula toluolica were vertically inherited. Furthermore, Desulfobacterium anilini and strain mXyS1 were identified, by dsrAB sequencing of 17 reference strains, as members of the donor lineage for those gram-positive Desulfotomaculum species which laterally acquired dsrAB.Dissimilatory (bi)sulfite reductase catalyzes the energy-generating step during the anaerobic respiration of sulfite or sulfate and thus represents a key enzyme of all sulfite-and sulfate-reducing prokaryotes (11,22,34). Recently, the genes encoding the alpha-and beta-subunits of this enzyme (dsrAB) have been used to infer the evolutionary history of dissimilatory (bi)sulfite reductases. For this purpose, a dsrAB database containing 75 entries for described sulfate-reducing prokaryotes (SRPs) (representing all known major evolutionary lineages of this guild) and four for sulfite-reducing microorganisms has been established (9, 12, 14-17, 23, 24, 28, 32, 33). Comparison of 16S rRNA-and DsrAB-based phylogenetic trees revealed congruent topologies for many SRP lineages, suggesting an ancient origin of dissimilatory (bi)sulfite reductase (33). This finding is consistent with isotopic evidence for biological sulfate reduction at 3.47 Gyr ago (31). However, we now recognize that the distribution of dsrAB among sulfatereducing species reflects a combination of divergence through speciation (vertical descent) and acquisition via lateral gene transfer from distantly related prokaryotes (15). The archaeal SRPs of the genus Archaeoglobus, the deep-branching thermophilic SRPs of the genus Thermodesulfobacterium, as well as a large number of thermophilic gram-positive Desulfotomaculum species, possess laterally-acquired (bi)sulfite reductases. In addition, the deltaproteobacterial SRP Desulfobacula toluolica was postulated to have laterally acquired its (bi)sulfite reductase relatively recently, since its dsrAB genes differed significantly from those of its close relatives, including Desulfobacter latus, which have vertically transmitted (bi)sulfite reductase genes. In the DsrAB tree, the putative DsrAB sequence of D. toluolica formed a well-supported monophyletic cluster with the laterally acquired DsrAB sequences of Desulfotomaculum species. Therefore, it was speculated that D. toluolica and the Desulfotomaculum species received their dsrAB from a common but so far unidentified deltaproteobacterial donor lineage (15). However, no information on transfer mechanism or donor lineages is available for any of the recognized dsrAB lateral gene transfer events.In an attempt to determine which additional genes might have been cotransferred with the dsrAB genes of D. toluolica and to reveal genetic traces indicative of the responsible transfer mechanism, the dsr operon (and flanking regions) of this SRP was sequenced. In a first step, a digoxigenin (DIG)-labeled 152-bp polynucleotide probe targeting dsrA was generated from D. toluolica DNA by using...

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A large cellulolytic enzyme (CelA) with the ability to hydrolyse microcrystalline cellulose was isolated from the extremely thermophilic, cellulolytic bacterium 'Anaerocellum thermophilum I . Full-length CelA and a truncated enzyme species designated CelA were purified to homogeneity from culture supernatants. CelA has an apparent molecular mass of 230 kDa. The enzyme exhibited significant activity towards Avicel and was most active towards soluble substrates such as CM-cellulose (CMC) and p-glucan. Maximal activity was observed between pH values of 5 and 6 and temperatures of 95 "C (CM-cellulase) and 85 "C (Avicelase). Cellobiose, glucose and minor amounts of cellotriose were observed as end-products of Avicel degradation. The CelAencoding gene was isolated from genomic DNA of 'A. thermophilum by PCR and the nucleotide sequence was determined. The celA gene encodes a protein of 1711 amino acids (190 kDa) starting with the sequence found at the Nterminus of CelA purified from 'A. thermophilum I . Sequence analysis revealed a multidomain structure consisting of two distinct catalytic domains homologous to glycosyl hydrolase families 9 and 48 and three domains homologous to family 111 cellulose-binding domain linked by Pro-Thr-Ser-rich regions. The enzyme is most closely related to CelA of Caldicellulosiruptor sacchamlyticus (sequence identities of 96 and 97% were found for the N-and C-terminal catalytic domains, respectively). Endoglucanase CelZ of Clostridium stercorarium shows 70.4% sequence identity to the N-terminal family 9 domain and exoglucanase CelY from the same organism has 69.2% amino acid identity with the C-terminal family 48 domain. Consistent with this similarity on the primary structure level, the 90 kDa truncated derivative CelA' containing the N-terminal half of CelA exhibited endoglucanase activity and bound t o microcrystalline cellulose. Due to the significantly enhanced Avicelase activity of full-length CelA, exoglucanase activity may be ascribed to the C-terminal family 48 catalytic domain.

BackgroundOne 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.ResultsThe 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 Clostridiumthermocellum and Clostridium stercorarium,Defluviitoga tunisiensis participated in the hydrolysis of hemicellulose producing ethanol, acetate, and H2/CO2. The latter three metabolites are substrates for hydrogentrophic and acetoclastic archaeal methanogenesis.ConclusionsObtained 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0581-3) contains supplementary material, which is available to authorized users.

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