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Dextran in Sugar Manufacture - Problem Evaluation and Enzyme-Based Mitigation

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Dextran in Sugar Manufacture - Problem Evaluation and Enzyme-Based Mitigation DEXTRAN IN SUGAR MANUFACTURE - PROBLEM EVALUATION AND ENZYME-BASED MITIGATION vorgelegt von M.Sc. vorgelegt von M. Sc. Karin Abraham von der Fakultät III – Prozesswissenschaften, der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Ingenieurwissenschaften - Dr.-Ing. - genehmigte Dissertation Promotionsausschuss: Vorsitzende: Prof. Dr.-Ing. habil. Cornelia Rauh Gutachter: Dr. Jan Maarten de Bruijn Gutachter: Prof. Dr.-Ing. Eckhard Flöter Gutachter: Dr. Lutz Popper Tag der wissenschaftlichen Aussprache: 20. Juni 2019 Berlin 2019 ABSTRACT It is well-known that the presence of the microbial polysaccharide dextran in sugar beet and cane juices can affect sugar manufacture in many ways. However, a controlled mitigation of dextran- induced effects during sugar processing by enzymatic decomposition is still not established practice, which is what this work is aiming at. The first step towards this is the detailed understanding of the effects of dextran as well as of enzymatically decomposed dextran during sugar manufacture. Therefore, laboratory juice purification and crystallisation experiments with synthetic thin and thick juices containing various dextran contents of different molecular masses were performed. This also includes enzymatic decompositions of dextran using various enzyme levels. Thereby, the most harmless reaction products with regard to these two process steps were identified. For the purification process by means of lime and carbonation gas, it was shown that dextran is involved in size and shape modifications of calcium carbonate particles precipitated during carbonation. This could affect both, the filtration as well as the purification performance. The data indicate that the presence of dextran with molecular masses above 10 kDa promotes calcium carbonate agglomeration. This was most pronounced for broadly distributed intermediate but rather low molecular mass dextran. Thus, a mixture of high and low molecular mass dextran fractions as well as mildly, insufficiently decomposed high molecular mass dextran caused the most dramatic increase in the size of calcium carbonate particles. A size-related evaluation of particle shape parameters has additionally revealed that particularly the shape of large-sized agglomerates was modified. The shape data indicate that the calcium carbonate agglomeration in dextran-free samples as well as in samples loaded with low molecular mass dextran is oriented. Once dextran of higher molecular mass was present (>85 kDa), the shape data suggest that the agglomeration was non- oriented. Viscosity measurements have additionally shown that the effect of the dextran contents relevant for raw juices in the beginning of sugar manufacture on the flow behaviour is minor. Thus, the major cause for impeded filtration performances as a part of beet raw juice purification can be attributed to the just mentioned particle size and shape modifications. Thereby, it is assumed that the filter cake porosity is modified due to the presence of more round agglomerates with smoother surfaces, resulting from non-oriented agglomeration. Similarly, the effects of different dextran fractions and enzymatically decomposed dextran on the size and shape distribution of sucrose crystals were investigated. The data from evaporative crystallisation experiments using synthetic thick juices indicate that three different crystal-shapes can be related to the presence of dextran, namely, cube-like, elongated as well as agglomerated crystals. The occurrence of these shapes seems to depend on the content of high molecular mass dextran, while low molecular mass dextran did not show a concentration dependency. For high contents of high molecular mass dextran and all contents of low molecular mass dextran, mainly an i increase in agglomerated crystals was found, accompanied by a rather low amount of elongated crystals. Here, again, it was found that mildly, insufficiently decomposed high molecular mass dextran still affects the sucrose crystal size and shape distribution, again negative effects were most pronounced for these broadly distributed intermediate but rather low molecular mass dextran fragments. Consequently, it has been shown that high as well as low molecular mass dextran affect the characteristics of calcium carbonate particles as well as of sucrose crystal in an undesirable way. Depending on the progress in enzymatic decomposition and thus the molecular mass of the reaction products, these dextran-induced effects could be mitigated. When decomposing dextran to reaction products with molecular masses of less than 10 kDa, no effects on the size and shape of calcium carbonate particles were found anymore. Besides, no dextran-related effects on the sucrose crystal characteristics were found once dextran was decomposed to reaction products with molecular masses below 5 kDa. A comprehensive study on the enzyme reaction on various dextran contents and initial molecular mass distributions was additionally done using size-exclusion and affinity chromatography. In doing so, it was found that, for all molecular mass distributions investigated, the molecular mass of dextran was gradually reduced with the increase in enzyme level and incubation time. However, the data also indicate that not only the total dextran content, but also the initial molecular mass distribution is decisive for the progress in enzymatic decomposition. Thus, for targeted dextranase dosage, the dextran content as well as its molecular mass distribution need to be known. To pay attention to this aspect as well, a practical tool for dextran analysis in sugar industrial practice was developed and benchmarked against the commonly used but rather inaccurate Haze Method. The method proposed is based on determining differences in optical rotation caused by the mechanical separation of dextran as a macromolecule from the juices by membrane filtration. Thereby, it was found that the presence of sucrose advantageously improved the separation of dextran via ultrafiltration. Separation efficiencies as well as specific cut-off values (10 kDa and 50 kDa) for the separation of dextran from aqueous sucrose solutions for two polyethersulfone membranes were determined. The data indicate that the basic membrane setting (specific cut-off of 10 kDa) enables the complete separation and therefore the analytical quantification of the whole molecular mass spectrum relevant for dextran-related effects. For higher dextran contents, a combination of both membranes additionally enables the differentiation between a high and a low molecular mass dextran fraction. Thus, the results indicate that the complete and also a fractionated dextran analysis in sugar cane and beet juices is possible with this new principle. For the first time, comprehensive basic scientific findings on the whole dextran issue have been combined to a basic framework for dextranase application in sugar industrial practice. ii ZUSAMMENFASSUNG Die Anwesenheit von mikrobiellen Polysacchariden in Zuckerrüben- sowie Zuckerrohrsäften kann zu diversen Beeinträchtigungen bei der Zuckergewinnung führen. Für gewöhnlich werden diese Dextran-induzierten Effekte über den Einsatz von Enzymen reduziert. Trotz jahrelangem Bestehen dieser Problematik, gibt es immer noch keinen kontrollierten Einsatz von Dextranasen in der Zuckerindustrie, dessen Ermöglichung sich diese Arbeit zum Ziel gesetzt hat. Dies setzt zunächst einmal das Verständnis der Prozesseffekte, die mit der Anwesenheit von Dextran und auch enzymatisch abgebautem Dextran einhergehen, voraus. Dafür wurden im Rahmen dieser Arbeit Saftreingungs- sowie Kristallisationsversuche im Labormaßstab mit synthetischen Dünn- und Dicksäften durchgeführt. Dabei wurden die Effekte von Dextran in Abhängigkeit der vorliegenden Konzentration und des Molekulargewichtes untersucht. Letzteres beinhaltet auch den enzymatischen Abbau von Dextran unter Verwendung verschiedener Enzymkonzentrationen. Als Resultat sollen für diese beiden bedeutenden Prozessschritte harmlose enzymatische Abbauprodukte und damit das Ziel des enzymatischen Abbaus identifiziert werden. Die Durchführung von Saftreinigungsversuchen nach dem Kalk-Kohlendioxid-Prinzip hat gezeigt, dass Dextran die Größe und die Gestalt der bei der Karbonatation ausgefällten Kalziumkarbonatpartikel modifiziert. Eine veränderte Partikelgröße und -gestalt kann die Filtration, aber auch den eigentlichen Reinigungseffekt beeinträchtigen. Die Daten haben gezeigt, dass die Anwesenheit von Dextran mit Molekulargewichten über 10 kDa die Kalziumkarbonat- Agglomeration fördert. Dies war insbesondere der Fall, wenn breit verteiltes, intermediäres niedermolekulares Dextran anwesend war. Schließlich war die Zunahme der Partikelfläche am stärksten ausgeprägt für eine Mischung aus der nieder- und hochmolekularen Fraktion aber auch für geringfügig und damit unzureichend enzymatisch abgebautes hochmolekulares Dextran. Eine größenabhängige Auswertung von Gestaltsparametern hat außerdem gezeigt, dass insbesondere die Gestalt von großen Agglomeraten durch Dextran verändert wird. Dabei deuten die Daten daraufhin, dass die Agglomeration von Kalziumkarbonat in Abwesenheit und in Anwesenheit von niedermolekularem Dextran orientiert erfolgt. Sobald Dextran mit höherem Molekulargewicht (>85 kDa) anwesend war, deuten die Daten auf eine nicht-orientierte Agglomeration hin. Es konnte außerdem gezeigt werden, dass der Effekt der für Rohsäfte relevanten Dextrankonzentrationen am Anfang der Zuckergewinnung auf die Viskosität eher
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