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Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with 13 C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of 13 C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than ␣-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.

This work evaluates the potential of vinasse (a waste obtained at the bottom of sugarcane ethanol distillation columns) as nutrient source for biohydrogen and volatile fatty acids production by means of anaerobic consortia. Two different media were proposed, using sugarcane juice or molasses as carbon source. The consortium LPBAH1 was selected for fermentation of vinasse supplemented with sugarcane juice, resulting in a higher H2 yield of 7.14 molH2 molsucrose(-1) and hydrogen content in biogas of approx. 31%, while consortium LPBAH2 resulted in 3.66 molH2/molsucrose and 32.7% hydrogen content in biogas. The proposed process showed a rational and economical use for vinasse, a mandatory byproduct of the renewable Brazilian energy matrix.

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The feasibility of the conversion of acetic acid, a metabolite commonly obtained during anaerobic fermentation processes, into oils using the yeast Cryptococcus curvatus was reported. This microorganism exhibited very slow growth rates on acetate as carbon source, which led to design a two-stage cultivation process. The first consisted of cell growth on glucose as carbon source until its complete exhaustion. The second step involved the use of acetate as carbon source under nitrogen limitation in order to induce lipid accumulation. A typical experiment performed in a bioreactor involved a preliminary yeast growth with a glucose initial concentration of 15 g/L glucose. Further additions of acetate and nitrogen source allowed a final lipid accumulation up to 50% (w/w). These promising results demonstrated the suitability of the technique proposed.

Ruminococcus albus is a Gram-positive rumen bacterium widely recognized for its high cellulolytic activity. It is the predominant cellulolytic bacterial species found in the rumen of cows [1], but is outnumbered by the other rumen cellulolytic species, R. flavefaciens and Fibrobacter succinogenes, in the rumen of sheep [2]. In vitro studies have shown that R. albus becomes predominant over the other two fibrolytic species in co-cultures on cellulose [3,4]. Data have also shown the negative interaction of R. albus and F. succinogenes on lucerne cell-wall polysaccharide degradation, as well as the complementary effect of R. albus and R. flavefaciens in lucerne hemicellulose degradation [5]. The interactions of cellulolytic species in fibre degradation therefore appear to be very complex, depending on several factors. An understanding of how the cellulolytic system of each species operates on natural substrates should aid in the determination of these complex interactions. The fibrolytic system of R. albus is composed of many different cellulases, xylanases Cellulose and wheat straw degradation by Ruminococcus albus was monitored using NMR spectroscopy. In situ solid-state 13 C-cross-polarization magic angle spinning NMR was used to monitor the modification of the composition and structure of cellulose and 13 C-enriched wheat straw during the growth of the bacterium on these substrates. In cellulose, amorphous regions were not preferentially degraded relative to crystalline areas by R. albus. Cellulose and hemicelluloses were also degraded at the same rate in wheat straw. Liquid state two-dimensional NMR experiments were used to analyse in detail the sugars released in the culture medium, and the integration of NMR signals enabled their quantification at various times of culture. The results showed glucose and cellodextrin accumulation in the medium of cellulose cultures; the cellodextrins were mainly cellotriose and accumulated to up to 2 mm after 4 days. In the wheat straw cultures, xylose was the main soluble sugar detected (1.4 mm); arabinose and glucose were also found, together with some oligosaccharides liberated from hemicellulose hydrolysis, but to a much lesser extent. No cellodextrins were detected. The results indicate that this strain of R. albus is unable to use glucose, xylose and arabinose for growth, but utilizes efficiently xylooligosaccharides. R. albus 20 appears to be less efficient than Fibrobacter succinogenes S85 for the degradation of wheat straw.

1D and 2D NMR experiments were used to analyse the synthesis of various metabolites by resting cells of Fibrobacter succinogenes S85 when incubated with [1-13 C]glucose, in both extracellular and cellular media. Besides the expected glycogen, succinate, acetate, glucose-1-P and glucose-6-P, maltodextrins and cellodextrins were detected. Maltodextrins were excreted into the external medium. They were found to have linear structures with a maximum degree of polymerization (DP) of about 6 or 7 units. Cellodextrins were located in the cells (cytoplasm and/or periplasm), and their DP was # 4. Both labelled (1-13 C and 6-13 C) and unlabelled maltodextrins and cellodextrins were detected, showing the contribution of carbohydrate cycling in F. succinogenes, including the reversal of glycolysis and the futile cycle of glycogen. The mechanisms of these oligosaccharide syntheses are discussed.

Cellulolytic bacteria play an important role in nature, borne out by the fact that microbial cellulose utilization is responsible for one of the largest material flows in the biosphere. Cellulolytic bacteria have thus been studied in detail, but, because of methodological difficulties in using solid cellulosic substrates, the majority of the studies were carried out on bacteria utilizing soluble substrates [1]. When data are available, it appears that most of the statements and concepts applicable to cellulolytic bacteria metabolizing soluble carbohydrates are not applicable to bacteria using cellulosic substrate [1]. In previous work, we showed that Fibrobacter succinogenes S85, a cellulolytic rumen bacterium, was able to synthesize and release oligosaccharides, identified by 2D-NMR techniques as maltodextrins (MD) and maltodextrin-1-phosphate (MD1P), upon incubation with glucose [2,3]. On the contrary, no cellodextrins were released by the bacteria. The synthesis of MD and MD1P was unexpected in a strain specialized in cellulose degradation and known to be unable to use maltose and starch [4]. These results prompted us to investigate whether such MD and MD1P were produced in bacteria metabolizing their natural substrate, cellulose. More precisely, the objectives were to answer the following questions (i) is the synthesis of MD and MD1P a specific feature of bacteria metabolizing glucose, or does it occur also on other carbohydrates, (ii) is F. succinogenes able to utilize these MDs as a carbon source, (iii) are cellodextrins produced when bacteria degrade cellulose, and (iv) are such observations physio- In this article we compared the metabolism of phosphorylated and unphosphorylated oligosaccharides (cellodextrins and maltodextrins) in Fibrobacter succinogenes S85 resting cells incubated with the following substrates: glucose; cellobiose; a mixture of glucose and cellobiose; and cellulose. Intracellular and extracellular media were analysed by 1 H-NMR and by TLC. The first important finding is that no cellodextrins were found to accumulate in the extracellular media of cells, regardless of the substrate; this contrasts to what is generally reported in the literature. The second finding of this work is that maltodextrins of degree of polymerization > 2 are synthesized regardless of the substrate, and can be used by the bacteria. Maltotriose plays a key role in this metabolism of maltodextrin. Maltodextrin-1-phosphate was detected in all the incubations, and a new metabolite, corresponding to a phosphorylated glucose derivative, was produced in the extracellular medium when cells were incubated with cellulose. The accumulation of these phosphorylated sugars increased with the degree of polymerization of the substrate. C; Glc1P, glucose-1-phosphate; Glc6P, glucose-6-phosphate; MD1P, maltodextrin-1-phosphate; MD, (malto-oligosaccharides) linear maltodextrins; MD t , the terminal Glc unit of MD; MD int , internal Glc units of MD and MD1P; TSP-d 4 , sodium 3-(trimethylsilyl)propionate-d 4 ; X, unknown glucose-1-ph...