Biomass pretreatment affects Ustilago maydis in producing itaconic acid

<p>Abstract</p> <p>Background</p> <p>In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus <it>Ustilago maydis </it>was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by <it>U. maydis</it>. Thus, this current study aims to first characterize <it>U. maydis </it>in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.</p> <p>Results</p> <p><it>U. maydis </it>was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, <it>U. maydis </it>was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.</p> <p>Conclusion</p> <p>The fungus <it>U. maydis </it>is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, <it>U. maydis </it>can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although <it>U. maydis </it>is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.</p

Practical screening of purified cellobiohydrolases and endoglucanases with α-cellulose and specification of hydrodynamics

<p>Abstract</p> <p>Background</p> <p>It is important to generate biofuels and society must be weaned from its dependency on fossil fuels. In order to produce biofuels, lignocellulose is pretreated and the resulting cellulose is hydrolyzed by cellulases such as cellobiohydrolases (CBH) and endoglucanases (EG). Until now, the biofuel industry has usually applied impractical celluloses to screen for cellulases capable of degrading naturally occurring, insoluble cellulose. This study investigates how these cellulases adsorb and hydrolyze insoluble α-cellulose − considered to be a more practical substrate which mimics the alkaline-pretreated biomass used in biorefineries. Moreover, this study investigates how hydrodynamics affects cellulase adsorption and activity onto α-cellulose.</p> <p>Results</p> <p>First, the cellulases CBH I, CBH II, EG I and EG II were purified from <it>Trichoderma reesei </it>and CBH I and EG I were utilized in order to study and model the adsorption isotherms (Langmuir) and kinetics (pseudo-first-order). Second, the adsorption kinetics and cellulase activities were studied under different hydrodynamic conditions, including liquid mixing and particle suspension. Third, in order to compare α-cellulose with three typically used celluloses, the exact cellulase activities towards all four substrates were measured.</p> <p>It was found that, using α-cellulose, the adsorption models fitted to the experimental data and yielded parameters comparable to those for filter paper. Moreover, it was determined that higher shaking frequencies clearly improved the adsorption of cellulases onto α-cellulose and thus bolstered their activity. Complete suspension of α-cellulose particles was the optimal operating condition in order to ensure efficient cellulase adsorption and activity. Finally, all four purified cellulases displayed comparable activities only on insoluble α-cellulose.</p> <p>Conclusions</p> <p>α-Cellulose is an excellent substrate to screen for CBHs and EGs. This current investigation shows in detail, for the first time, the adsorption of purified cellulases onto α-cellulose, the effect of hydrodynamics on cellulase adsorption and the correlation between the adsorption and the activity of cellulases at different hydrodynamic conditions. Complete suspension of the substrate has to be ensured in order to optimize the cellulase attack. In the future, screenings should be conducted with α-cellulose so that proper cellulases are selected to best hydrolyze the real alkaline-pretreated biomass used in biorefineries.</p

How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis

<p>Abstract</p> <p>Background</p> <p>In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus <it>Trichoderma reesei</it>. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis.</p> <p>Results</p> <p>After expression in the yeast <it>Kluyveromyces lactis</it>, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from <it>T. reesei</it>. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin-induced reduction in particle size and crystallinity resulted in high cellulose hydrolysis rates.</p> <p>Conclusions</p> <p>Recombinant swollenin can be easily produced with the robust yeast <it>K. lactis</it>. Moreover, swollenin induces deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization). For the first time, this study quantifies and elucidates in detail how swollenin affects different cellulosic substrates and their hydrolysis.</p

Модернизация системы управления частотой вращения турбины автономного преобразователя энергии

В работе рассматривается модернизация существующей системы управления частотой вращения турбины автономного преобразователя энергии. Результатом работы является реализованная модель управления, установленная в преобразователь энергии. Работа выполняется на базе ОАО ТЭМЗ.The paper deals with the modernization of the existing control system of the turbine speed of the autonomous energy converter. The result is a realized control model installed in the energy converter. The work is done on the basis of OJSC TEMZ

IL4I1 Is a Metabolic Immune Checkpoint that Activates the AHR and Promotes Tumor Progression

Immune checkpoint blockade therapy induces the AHR-activating enzyme IL4I1, which promotes tumor progression, through its effects on tumor cell motility and adaptive immunity

Biocatalytic conversion of cellulose towards itaconic acid

Naturally occurring cellulose can be used as a renewable resource for the sustainable production of platform chemicals such as itaconic acid. The biocatalytic conversion of cellulose is a very promising approach due to its high selectivity, mild conditions, and low exergy loss. However, such biocatalytic processes are still seldom applied at the industrial scale since the single conversion steps (pretreatment, hydrolysis, and fermentation) exhibit low efficiencies or high costs. To allow a knowledge-based optimization, each step was analyzed in detail within this thesis. Thereby, new (integrated) approaches and screening technologies were also established. Within this thesis, a detailed understanding of the biocatalytic conversion of cellulose to itaconic acid was generated. Not only all essential conversion steps were investigated in detail, but also novel (integrated) approaches as well as technologies were developed. In the future, the conversion steps need to be further harmonized, and (in-situ) product recovery should be implemented, as well as the recycling of water, cellulases, microorganisms, and unconverted cellulose. Moreover, cellulases and microorganisms need to be improved and adapted to integrated process configuration such as SSF. In conclusion, the results presented within this thesis are the fundamental basis for a further knowledge-based improvement and pave the way for an economically feasible production process converting cellulose to itaconic acid