Browsing by Subject "Lignocellulose"

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Bioethanol production from lignocellulosic biomass
(2023) Hoppert, Luis; Kölling, Ralf
The aim of this thesis was to develop a high gravity second-generation bioethanol process and investigate the effects of a high solid loading. The insights gained from the initial experiments helped to understand the underlying mechanism behind the limitations of a high solid loading. Based on these findings, strategies were developed to overcome these limitations.
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.
Continuous synthesis of 5‐hydroxymethylfurfural from biomass in on‐farm biorefinery
(2022) Świątek, Katarzyna; Olszewski, Maciej P.; Kruse, Andrea
5‐hydroxymethylfurfural (HMF) is the object of extensive research in recent times. The challenge in the industrial production of HMF is the choice of cheap, hexose feedstock. This study compares continuous HMF synthesis from hexoses—fructose and glucose, and biomass—Miscanthus × giganteus and chicory roots. The experiments were conducted in technical‐scale biorefinery (TRL 6/7). In the first stage, optimal conditions for the production of HMF from hexoses were selected using sulfuric acid as a catalyst in an aqueous medium. The following conditions were chosen for fructose: temperature of 200°C, the reaction time of 18 min, and pH = 2, and for glucose: 210°C, 18 min, and pH = 3. Under these conditions, the HMF yield was 56.5 mol% (39.6 wt.%) from fructose and 18.1 mol% (12.6 wt.%) from glucose. From the biomass, the HMF yields were 36.7 and 16.2 wt.% for miscanthus and chicory roots, respectively. Some results from the conversion of biomass solutions are unexpected and show a need for further investigations. This work has demonstrated the capacity to produce HMF from biomass as part of an environmentally friendly process in a biorefinery. Further research in this field and process optimization will be a step forward in the sustainable production of bioplastics.
Evaluation of bio-oil produced from fast pyrolysis of lignocellulosic biomass as carbon source for bacterial bioconversion
(2020) Arnold, Stefanie; Hausmann, Rudolf
Scarcity of fossil resources, climate change and growing world population demand the transition from a fossil-based economy towards a bioeconomy – a knowledge-based strategy which relies on the efficient and sustainable integration of bio-based resources into value-added process chains. As lignocellulosic biomass is an abundant renewable resource which does not directly compete with food and feed, its deployment in biorefineries is of special interest for a sustainable bioeconomy. Owing to its compact and complex structure, suitable conversion techniques need to be selected. Combinations of thermochemical and biochemical conversion technologies are considered to be a promising approach regarding a fast and efficient conversion of lignocellulosic biomass into value-added products. Bio-oil derived from fast pyrolysis of lignocellulosic biomass is a complex mixture and composed of water and a wide variety of organic components. Among these components pyrolytic sugars and small organic acids are particularly interesting as potential carbon sources for microbial processes. However, bio-oil also comprises many unidentified substances, as well as components which are known to display adverse effects on microbial growth. To evaluate the potential and challenges of bio-oil as an alternative and sustainable carbon source for bacterial bioconversion this thesis was divided into three parts (Figure 1). In Part I different pretreatment strategies were applied and evaluated regarding their effect on stability and detoxification of bio-oil fractions. For this purpose, the organic solvent tolerant bacterial strain Pseudomonas putida KT2440 was applied as a reference system and cultivated on different pretreated bio-oil fractions. It was shown that solid phase extraction is a suitable tool to obtain bio-oil fractions with significantly increased stability along with less inhibitory substances. Part II is focused on the evaluation of small organic acids mainly present in bio-oil with respect to their suitability as feedstock for bacterial growth. Four biotechnological production hosts Escherchia coli, Pseudomonas putida, Bacillus subtilis and Corynebacterium glutamicum were cultivated on different concentrations of acetate, mixtures of small organic acids, as well as pretreated bio-oil fractions as carbon source for their growth. Results reveal that P. putida, as well as C. glutamicum metabolizes acetate – the major small organic acid generated during fast pyrolysis of lignocellulosic biomass – as sole carbon source over a wide concentration range and grow on mixtures of small organic acids present in bio-oil. Moreover, both strains show a distinct potential to tolerate inhibitory substances within bio-oil. Part III describes the growth behavior of a genetically engineered, nonpathogenic bacterium Pseudomonas putida KT2440 and its simultaneous heterologous production of rhamnolipid biosurfactants on bio-oil derived small organic acids and pretreated fractions. Results suggest that both maximum achievable productivities and substrate-to-biomass yields are in a comparable range for glucose, acetate, as well as the mixture of acetate, formate and propionate. Similar yields were obtained for a pretreated bio-oil fraction, although with significantly lower titers. In conclusion, this thesis shows that microbial valorization of bio-oil is a challenging task due to its highly complex and variable composition, as well as its adverse effects on microbial growth and issues with analytical procedures. This work depicts a proof of concept by outlining a potential biorefinery route for microbial valorization of pretreated bio-oil and its unexploited side streams. It provides a step in search of suitable bacterial strains for bioconversion of lignocellulosicbased feedstocks into value-added products and thus contributes to establishing bioprocesses within a future bioeconomy.
Verbesserung der Energie-, Stoff- und Emissionsbilanzen bei der Bioethanolproduktion aus nachwachsenden Rohstoffen
(2010) Fleischer, Sven; Senn, Thomas
In this thesis, a process was realized that uses starchy raw material (triticale) as well as lignocellulosic biomass (corn silage) in one ethanol production process. In contrast to other so called 2nd generation ethanol processes, which only use lignocellulosic material, the problem of the very low potential ethanol concentration (