Partner und Internationale Organisationen
(Englisch)
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DK, F, D, H, I, NL, P, E, S, CH, TR, GB
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Abstract
(Englisch)
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This work describes the metabolism of Clostridium thermolacticum cultivated on lactose and the influence of hydrogen, generated during fermentation, on the acetate productivity. In order to study the metabolism of this species and understand how the products are generated, a continuous bioreactor system operated under anaerobic conditions has been set up. In continuous cultures of C. thermolacticum degrading lactose, besides acetate, by-products including ethanol, lactate, glucose, galactose, carbon dioxide and hydrogen were formed. Acetate, ethanol, hydrogen and carbon dioxide were primary metabolites, while lactate, glucose and galactose were non-growth associated. The lactose metabolism of C. thermolacticum was unravelled: intracellular concentration of cofactors NAD+, NADH, ATP, ADP and the activity of several key-enzymes were determined. Consequently, the pathway for product formation was proposed. Hydrogen was the major gas generated: a maximum hydrogen productivity of 3.00 mmol l-1 h-1 was achieved under slightly alkaline conditions. To test the effect of hydrogen partial pressure on the metabolism of lactose and on the acetate productivity, the efficient hydrogen-consuming species Methanothermobacter thermoautotrophicus (methanogenic Archaea) has been added to the continuous culture of C. thermolacticum. As a consequence, in this coculture, the hydrogen partial pressure was noticeably decreased and methane was the major gas generated. Acetate became the major product of the liquid phase, ethanol concentration was dramatically reduced, but lactate was still produced at low dilution rates. Moorella thermoautotrophica (homoacetogenic bacteria) was added to the coculture to convert lactate into acetate. As a result, acetate was almost the sole organic product of the liquid phase. The maximal acetate volumetric productivity was 3.9 mmol l-1 h-1, obtained in this consortium at dilution rate 0.058 h-1. The yield coefficient of hydrogen on lactose when C. thermolacticum was cultivated in the coculture or in the consortium was 5.2 mol mol-1, while only 2.5 mol mol-1 in pure culture. This yield, two-times higher in coculture and in consortium indicates that, in the presence of hydrogenotrophs that maintain the hydrogen partial pressure at low levels, a larger flux of redox mediators (NADH, ferredoxin) was directed towards hydrogen production instead of ethanol formation, in C. thermolacticum. Based on hydrogen partial pressure measurements and Gibbs free energy calculations it was demonstrated that hydrogen production was inhibited by high hydrogen partial pressure. The co-cultivation is advantageous for C. thermolacticum from an energetic aspect: more ATP is generated due to increased acetogenesis that causes increased biomass formation and allows higher lactose consumption rate and increased acetate productivity. Based on Gibbs free energy calculations, it appears that the production of acetate and methane from lactose in coculture and consortium is thermodynamically more attractive than a mixture of acetate, ethanol, lactate, carbon dioxide and hydrogen, in pure culture. In the consortium, through efficient in situ hydrogen scavenging, the metabolic pattern of C. thermolacticum was modified in favour of acetate production, instead of reduced by-products. In this work we established the general strategy for metabolite production by heterofermentative bacterium. By co-cultivation, adding two other appropriate strains, the metabolism of the heterofermentative bacterium was redirected to increase the yield and the productivity of the desired product. On the basis of results obtained, a process using lactose present in milk or whey permeate could be developed for the production of acetate.
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