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On-line monitoring devices for the precise determination of a multitude of components are a prerequisite for fast bioprocess quantification. On-line measured values have to be checked for quality and consistency, in order to extract quantitative information from these data. In the present study we characterized a novel on-line sampling and analysis device comprising an automatic photometric robot. We connected this on-line device to a bioreactor and concomitantly measured six components (i.e. glucose, glycerol, ethanol, acetate, phosphate and ammonium) during different batch cultivations of Pichia pastoris. The on-line measured data did not show significant deviations from off-line taken samples and were consequently used for incremental rate and yield calculations. In this respect we highlighted the importance of data quality and discussed the phenomenon of error propagation. On-line calculated rates and yields depicted the physiological responses of the P. pastoris cells in unlimited and limited cultures. A more detailed analysis of the physiological state was possible by considering the off-line determined biomass dry weight and the calculation of specific rates. Here we present a novel device for on-line monitoring of bioprocesses, which ensures high data quality in real-time and therefore refers to a valuable tool for Process Analytical Technology (PAT).

AbstractMixed substrate feeding strategies are frequently investigated to enhance the productivity of recombinant Pichia pastoris processes. For this purpose, numerous fed batch experiments or time‐consuming continuous cultivations are required to optimize control parameters such as the substrate mixing ratio and the applied methanol concentration. In this study, we decoupled the feeding of methanol and glycerol in a mixed substrate fed batch environment to gain process understanding for a recombinant P. pastoris Muts strain producing the model enzyme horseradish peroxidase. Specific substrate uptake rates (qs) were controlled separately, and a stepwise increased qGly‐control scheme was applied to investigate the effect of various substrate fluxes on the culture. The qs‐controlled strategy allowed a parallel characterization of the metabolism and the recombinant protein expression in a fed batch environment. A critical‐specific glycerol uptake rate was determined, where a decline of the specific productivity occurred, and a time‐dependent acceleration of protein expression was characterized with the dynamic fed batch approach. Based on the observations on recombinant protein expression, propositions for an optimal feeding design to target maximal productivities were stated. Thus, the dynamic fed batch strategy was found to be a valuable tool for both process understanding and optimization of product formation for P. pastoris in a mixed substrate environment. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012

This work investigates the influence of the positive regulator XYR1 of Trichoderma harzianum on the production of cellulolytic enzymes, using sugarcane bagasse as carbon source. Constitutive expression of xyr1 was achieved under the control of the strong Trichoderma reesei pki1 promoter. Five clones with xyr1 overexpression achieved higher xyr1 expression and greater enzymatic productivity when cultivated under submerged fermentation, hence validating the genetic construction for T. harzianum. Clone 5 presented a relative expression of xyr1 26-fold higher than the parent strain and exhibited 66, 37, and 36% higher values for filter paper activity, xylanase activity, and β-glucosidase activity, respectively, during cultivation in a stirred-tank bioreactor. The overexpression of xyr1 in T. harzianum resulted in an enzymatic complex with significantly improved performance in sugarcane bagasse saccharification, with an enhancement of 25% in the first 24h. Our results also show that constitutive overexpression of xyr1 leads to the induction of several important players in biomass degradation at early (24h) and also late (48h) timepoints of inoculation. However, we also observed that the carbon catabolite repressor CRE1 was upregulated in xyr1 overexpression mutants. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression and suggest an attractive approach for increasing total cellulase productivity in T. harzianum.

The concentrations of glucose and ethanol in substrates from bioethanol processes have been modeled by near infrared (NIR) spectroscopy data. NIR spectra were acquired in the wavelength range of 1100-2300 nm by means of a transflectance probe for measurements in liquid samples. For building of regression models a genetic algorithm has been applied for variable selection, and partial least-squares (PLS) regression for creation of linear models. A realistic estimation of the prediction performance of the models was obtained by a repeated double cross-validation (rdCV). Reduced data sets with only 15 variables showed improved prediction qualities, in comparison with models containing 235 variables, particularly for the determination of the ethanol concentration in distillation residues (stillages). The squared correlation coefficient, R(2), between the concentrations obtained by HPLC analysis and the concentrations derived from NIR data (using 15 selected wavelengths, test set samples) was 0.999 for ethanol in stillage, and 0.977 for glucose in mash. The standard deviation of prediction errors, SEP, obtained from test set samples was 0.6 g L(-1) for ethanol (2% of the mean ethanol concentration), and 2.0 g L(-1) for glucose (9.6% of the mean glucose concentration).

The binary oxide Bi26Mo10O69 is the parent compound of a new family of oxygen ion conductors with column type structure. High anion conductivity is observed along the [Bi12O14] columns within the surrounding MoO4 tetrahedra. High conductivity at intermediate temperatures is an important property but the electrochemical stability of the materials plays also an important role for future applications. Since bismuth-based electrolytes turned out to be not stable enough for an application in fuel cells, it is to be assumed that these materials are also easily decomposed. In this work, the decomposition limit was investigated by a voltammetric technique using very small samples. The results show a stability limit located approximately 100 mV more positive than in BIMEVOX. This is probably related to the reduction of Mo(VI) to Mo(IV). No change of oxygen stoichiometry was observed in the Bi26Mo10O69-based material in the course of a voltammogram before the primary reduction process occurs.

A model for steam gasification of biomass was developed by applying thermodynamic equilibrium calculations. With this model, the simulation of a decentralized combined heat and power station based on a dual fluidized-bed steam gasifier was carried out. Fuel composition (ultimate analysis and moisture content) and the operating parameters, temperature and amount of gasification agent, were varied over a wide range. Their influences on amount, composition, and heating value of product gas and process efficiencies were evaluated. It was shown that the accuracy of an equilibrium model for the gas composition is sufficient for thermodynamic considerations. Net electric efficiency of about 20% can be expected with a rather simple process. Sensitivity analysis showed that gasification temperature and fuel oxygen content were the most significant parameters determining the chemical efficiency of the gasification.

Lignocellulosic plant biomass is the world's most abundant carbon source and has consequently attracted attention as a renewable resource for production of biofuels and commodity chemicals. Still the process is economically not fit enough to compete with then use of fossil resources, and the costs associated with enzymatic hydrolysis and product recovery are the major obstacle. The discovery of the role of non-hydrolytic enzymes in lignocellulose hydrolysis has recently contributed significant improvements to hydrolysis but also added new challenges to the biomass to ethanol process. Transfer of the new insights to the industrial scale and shaping the enzymes to tolerate associated adverse conditions has now shown first success, thus optimizing the economy of cellulosic ethanol (or other biofuel) production.

Trichoderma harzianum has attracting attention for its potential alternative use in biofuel production, due to a recognized competence for high diversity glycoside hydrolases (GH) enzyme complex, including higher β-glucosidases and auxiliary proteins, using low-cost carbon sources. This strain constitutively overexpressed the global regulator putative methyltransferase - LAE1, in order to improve the GHs production. The recombinant strain achieved 79-fold increase in lae1 expression and high GHs productivity. The evaluation of the LAE1 impact to induce the GHs used soluble and lignocellulose inexpensive carbon sources in a stirred-tank bioreactor. Using sugarcane bagasse with sucrose, the overexpression of lae1 resulted in significantly increment of gh61b (31x), cel7a (25x), bgl1(20x) and xyn3 (20x) genes expression. Reducing sugar released from pretreated sugarcane bagasse, which hydrolyzed by recombinant crude enzyme cocktail, achieved 41% improvement. Therefore, lae1 overexpression effectively is a promising improving GHs target for biomass degradation by T. harzianum.