Browsing by Subject "Bioraffinerie"

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Biotechnological conversion of lignocellulose hydrolyzates : model microorganisms for a bio-based economy
(2020) Horlamus, Felix; Hausmann, Rudolf
Lignocellulose has substantial potential as a carbon source in a bio-based economy. It is the most abundant renewable raw material on earth and is available in large quantities as waste from the agriculture, food and wood industry. It is composed mainly of the polymers lignin, cellulose and hemicellulose. In contrast to glucose derived from cellulose, hemicellulose sugars often remain unused although 60 billion tons of hemicelluloses are produced annually. Hemicelluloses are a group of heterogeneous polysaccharides consisting of different monomers such as D xylose, D arabinose, D mannose and D galactose. Lignocellulose is mostly depolymerized in order to obtain fermentable sugars. During the depolymerization process, inhibitors such as organic acids or furan aldehydes can be formed or released, which could be problematical for biotechnological processes. The aim of this thesis was to develop and evaluate bacterial-based biotechnological processes capable of using hemicellulose sugars as a source of carbon. First, Pseudomonas putida KT2440 was chosen. Pseudomonades are claimed as a promising chassis in biotechnology due to their versatile and robust metabolism. Unlike other Pseudomonades, the strain KT2440 is classified as biosafety level 1 in the American Type Culture Collection (ATCC). However, these bacteria can metabolize glucose as the only lignocellulose monosaccharide. Cellvibrio japonicus was the second selected bacterium. This strain is not yet established as a microbial host in biotechnology, but can degrade a huge portfolio of plant cell wall polysaccharides and is also classified as biosafety level 1 in ATCC. The topic of the first publication was to engineer P. putida KT2440 strains for metabolizing the hemicellulose monosaccharides xylose and arabinose and characterize their growth behavior. Initially, an arabinose metabolizing strain with the araBAD operon and a xylose metabolizing strain with xylAB operon was constructed. Later on, these strains were cultivated in minimal salt medium with glucose, xylose and arabinose as carbon sources in Erlenmeyer flasks. The recombinant P. putida KT2440 strains metabolized xylose and arabinose with high growth rates comparable to glucose. It turned out that both engineered strains were able to grow on both pentoses as well as on mixtures of glucose xylose and arabinose. The intent of the second publication was to evaluate P. putida KT2440 as a platform model organism for bioconversion of lignocellulose hydrolyzates. Strains were cultivated in minimal salt medium with several hydrolyzates as carbon source in Erlenmeyer flask and bioreactor. In addition, the growth-inhibiting effect of major toxic substances contained in lignocellulose hydrolyzates on P. putida KT2440 was analyzed via cultivation experiments. Several suitable hydrolyzates were figured out for this strain. Formic acid and acetic acid proved to be relatively unproblematic under pH neutral conditions, whereas furfural and hydroxymethylfurfural (HMF) had a negative effect on the bacterial growth. A diauxic-like growth behavior was revealed via fed batch bioreactor cultivations, since pentoses were almost not consumed with sufficient glucose supply. Consequently, feed-medium was added step-by-step in the next experiment. The applied feed profile did lead to an almost complete metabolization of xylose. The purpose of the third publication was to evaluate C. japonicus as a potential host strain for the one‐step bioconversion of xylans into rhamnolipids. Cultivation experiments were performed in Erlenmeyer flasks filled with minimal salt medium and containing different carbon sources. Furthermore, the strain was transformed with the plasmid pSynPro8oT carrying rhlA (encodes acetyltransferase) and rhlB (encodes rhamnosyltransferase I) to complete the rhamnolipid metabolism. The strain grew on all main lignocellulose monosaccharides as well as, on different xylans. Mono rhamnolipids were produced with the engineered strain using xylans as carbon source. This is particularly interesting as most industrially relevant bacteria are not able to depolymerize wood polymers. As the product yields were quite low, there are still many challenges in order to achieve an economically efficient process. Nevertheless, to the best of our knowledge, it is the first published one step bioconversion of hemicellulose polymers into rhamnolipids. In total, P. putida KT2440 turned out as a flexible and powerful model organism and two xylose and arabinose metabolizing strains were constructed. Moreover, bioreactor cultivations with lignocellulose hydrolyzates were performed and a feeding strategy to overcome diauxic-like growth behavior was presented. A proof of concept for a one-step bioconversion of xylans into rhamnolipids with a recombinant C. japonicus strain was successfully demonstrated.
Production of lactic acid and methane from renewable resources : an innovative green biorefinery concept for biogas process chains
(2015) Haag, Nicola Leonard; Jungbluth, Thomas
The increasing demands of world’s growing population for food, energy and products, the effects of climate change and the depletion of fossil resources forces the development of sustainable industries. Based on renewable resources, state-of-the-art processes have to be transformed to eco-friendly production sequences to lead the industry to a new, bio-based economy. An essential part of the bio-based economy will be biorefineries, as they enable the production of goods and energy from bio-based resources. The aim of this study was to establish an innovative green biorefinery concept to optimize biogas process chains. The green biorefinery concept was set up to both utilize and add value to green biomass, as well as other common raw substrates used in biogas processing, by producing platform chemicals and biogas. New ensiling techniques were applied, in order to increase the amount of valuable ingredients in the silage with a special focus on lactic acid. After solid-liquid separation of the silage to exploit organic acids, the solid residue was used for anaerobic digestion. In particular the objectives were: (1) to clarify which valuable chemicals can be increased in significant amounts, depending on the raw substrate, (2) to examine the technical, chemical and biological parameters affecting the increase of valuable products in the silage and (3) to investigate the methane formation potential of the residual biomass and the fresh silage to identify potential methane losses. Lactic acid was the most promising chemical, increased to highest amounts during the ensiling process. The addition of carbonated lime was the most effective treatment to increase the amount of lactic acid, requiring a high fermentability coefficient of the utilized raw substrate. Additional lactic acid producing bacteria can help to stabilize the silage and promote the growth of lactic acid contents. Supplying the lactic acid bacteria with additional trace elements (manganese) showed no effect on lactic acid production. The comparison of specific methane yields of the fresh silages with the corresponding solid residues always yielded higher values for the fresh silage (not always significant), due to the loss of volatile solids during the fractionating. Furthermore, there is a loss of overall methane production, due to the reduction of mass while fractionating. An initial economic assessment revealed that selected variations of the treated raw substrates maize and grass offer a huge potential for the presented biorefinery concept, as the increase in lactic acid contents was immense while simultaneously having no significant losses in specific methane yields. Crucial importance for the economic feasibility lies on the downstream process of lactic acid. Future research has to be focused on establishing adequate extraction techniques, as the extraction and purity of lactic acid is the primary challenge for the economic viability of the concept. In the context of adding value to existing biogas process chains, the presented green biorefinery concept is an alternative conversion path of biomass and will likely be of monetary interest in the near future. Moreover, the improved silages can be beneficial in other applications, such as the production of middle chain fatty acids for further processing. The presented biorefinery concept is of high value for numerous applications and shows an improved method of green biorefining, which can contribute to leading our society and industry to a sustainable and multifaceted future.