Xylose metabolism in Bacteroides xylanolyticus X5-1

Plant cell walls represent a major part of the available biomass on earth. They are mainly composed of the energy-rich polymers lignin, cellulose, and hemicellulose. For many decades, research is done to exploit agricultural and forestry wastes as renewable resources. Much research was focused on the degradation of cellulose. In contrast, hemicellulose. has got less attention, though it can account for up to 40% of the total dry weight of plant cell walls. Fermentation by anaerobic bacteria offers the possibility to conserve most energy fixed in the energy-rich polymeric and monomeric sugars in the form of organic acids and solvents (e.g. acetic acid, butanol and acetone).A project in which the anaerobic conversion of hemicellulose to potentially biotechnological interesting products was investigated, was divided into two parts. One part, performed by Philippe Schyns, concerned the microbial degradation of xylan, which was used as a model substrate for hemicellulose. Several xylanolytic enzymes (endo-xylanase, β-xylosidase, acetylesterase, α-L-arabinofuranosidase) were purified and characterized. The mode of action of some of these enzymes was investigated. Furthermore, the induction mechanism of xylanase and β-xylosidase was studied. The results of this research will be presented in a separate thesis. The other part of the project, of which the outcomes are given in this thesis, was focused mainly on the fermentation of xylose, a major constituent of hemicelluloses.Bacteroides xylanolyticus X5-1 was used as a model organism. This organism had been isolated from fermenting cattle manure. B. xylanolyticus X5-1 can only grow on one specific hemicellulose, xylan. Cellulose and other hemicelluloses could not be utilized for growth. This fact made the organism interesting for studying the (regulation of the) xylanolytic enzyme synthesis, since interferences from other (hemi)cellulolytic enzymes could be excluded. In addition, the organism could ferment a wide variety of monomeric sugars, produced a mixture of end products, and showed a relatively high growth rate. These latter features made B. xylanolyticus X5-1 a suitable microorganism for studying the regulation of the anaerobic xylose fermentation.Information concerning the composition and degradation of biomass, and the (regulation of) product formation from biomass has been reviewed in a general context in chapter 1. Some biotechnological applications of biomass fermentation have been mentioned in this chapter as well.Using 14C-labelled xylose, the xylose uptake system of this organism was studied. It was shown that xylose transport occurs via an active uptake system, and probably a binding protein was involved. The exact mechanism of xylose uptake remains to be elucidated. Based on mass balance calculations, measuring specific enzyme activities of key enzymes of catabolic pathways, and determining label distribution patterns with 13C-NMR, the pentose phosphate pathway in conjunction with the glycolysis was shown to be operative in xylose fermentation by B. xylanolyticus X5-1. Acetate, ethanol H 2 , CO 2 and formate were the main end products formed during xylose metabolism. At higher xylose concentrations, lactate and 1,2-propanediol were produced in small amounts as additional products. Reducing equivalents formed during the oxidation of glyceraldehyde-3-PO 4 and pyruvate, were used for the production of H 2 , formate, and ethanol. According to the proposed pathway about 2.5 mol of ATP, synthesized at substrate level, were generated per mol of xylose degraded. This part of the research is presented in chapter 2.The degradation of mixtures of hexoses and pentoses by B. xylanolyticus X5-1 is described in chapter 3. Batch culture cells did not show diauxic growth or a substrate preference for either glucose, xylose, arabinose or rhamnose, independent of the substrate the organism was grown on. In contrast, glucoselimited continuous culture cells were not able to consume xylose, unless some glucose or pyruvate was present as additional substrate. Glucose-limited continuous culture cells exhibited low activities of xylose transport and of xylose isomerase. Xylulose kinase could not be detected at all. Upon addition of xylose as single substrate to the glucose grown cells no increase in the transport rate and the isomerase and kinase activities was observed. However, when together with the xylose some glucose was added, all activities were induced. In the presence of chloramphenicol, an inhibitor of protein synthesis, xylose isomerase and xylulose kinase were not induced. The transport activity increased in a similar fashion as in the absence of chloramphenicol, suggesting that the transport system had to be activated and not induced. These experiments showed that i) xylose isomerase and xylulose kinase were regulated at the level of protein synthesis, ii) xylose transport was constitutively present, and iii) apparently, the glucose grown cells were carbon and energy limited. When grown under non-limiting conditions, as will probably happen in hemicellulose hydrolysates, B. xylanolyticus X5-1 can use sugar mixtures. This certainly is of biotechnological relevance, as conversion of the major substrate xylose will not be negatively affected by the minor, often preferred substrate glucose.Chapter 4 describes the effect of a low partial hydrogen pressure on the xylose metabolism in B. xylanolyticus X5-1. When grown in pure culture in the chemostat with xylose as the growth limiting substrate, B. xylanolyticus X5-1 produced acetate, ethanol, H 2 and CO 2 as the only end products. When grown in the presence of the methanogen Methanospirillum hungatei JF-1, xylose was converted to mainly acetate and CO 2 and presumably H 2 . Due to the cocultivation an increased biomass production was observed. H 2 could hardly be detected because it was efficiently converted to CH 4 by the methanogen. Ethanol was no longer produced. This type of regulation of product formation has been observed in many anaerobic microorganisms. However, xylose fermentation in B. xylanolyticus X5-1 was not only regulated at product level, but also on enzyme level. In cell free extracts of the pure culture of B. xylanolyticus X5-1 NAD and NADP-linked acetaldehyde and ethanol dehydrogenases could be detected. When grown in mixed culture with M.hungatei JF-1 these enzymes were no longer observed. The NAD and NADP-linked dehydrogenases were induced sequentially, when the interspecies electron transfer was inhibited, unless chloramphenicol was present. These results showed that product formation at low partial hydrogen pressure in B. xylanolyticus X5-1 is regulated at the level of enzyme synthesis.Several environmental conditions were used to affect xylose metabolism of B. xylanolyticus X5-1 ( chapter 5 ). Growth under a hydrogen atmosphere did not affect the xylose metabolism significantly. CO inhibited H 2 production from xylose completely with formate and ethanol as major reduced products. An increased ethanol yield resulted in a reduced amount of acetate and biomass formation. Xylose metabolism could also be affected by using alternative electron acceptors such as acetol, acetone, acetoin, and dihydroxy acetone. They were reduced to their corresponding alcohols 1,2-propanediol, 2-propanol, 2,3-butanediol, and glycerol, respectively. With these electron acceptors mainly acetate and CO 2 were formed and hardly any H 2 , formate and ethanol. As a result of more acetate formation, biomass production increased. In continuous culture with xylose as growth limiting substrate and acetol as electron acceptor, product formation from xylose shifted to mainly acetate and CO 2 as well. Acetol was not only reduced to 1,2-propanediol, but also converted to acetone. In gel activity staining of the alcohol dehydrogenases revealed that i) the NADP-linked ethanol dehydrogenase was repressed in the xylose + acetol grown culture, ii) the NADP-linked ethanol dehydrogenase in the xylose grown cells exhibited a nonspecific activity for both ethanol and 1,2-propanediol, and iii) another, also NADP-linked, 1,2-propanediol dehydrogenase was induced in the xylose + acetol grown cells.The data presented in this thesis show that it is possible to modulate the xylose metabolism of B. xylanolyticus X5-1 by several methods and at different levels during metabolism. The outcomes of this research might be applicable for other microorganisms of biotechnological value as well. Accordingly, the results can be used for biotechnological production processes and the biotechnological formation of valuable products (e.g. microbiological reduction processes, optically active products, enzymes like (stereospecific) alcohol dehydrogenases).