Influence of oxygen transfer rate and media composition on fermentation ofd-xylose byPichia stipitis NRRL Y-7124

Summary The optimization of ethanol production byPichia stipitis NRRL Y-7124 was analysed by ATP balance. Ethanol volumetric productivity was maximal (0.5–0.6 g/l h) only over a narrow range of oxygen transfer rates (3–5 mmol O₂/l h). Trace element supplements increased ethanol volumetric productivity 20%. Biotin and thiamine did not significantly affect ethanol yield. Vitamins and trace elements were not synergistic. Organic nitrogen source from yeast extract was used for growth simultaneously to ammonia.     

Influence of magnesium concentration on growth,ethanol and xylitol production by Pichia stipitis NRRL Y-7124

Summary The effect of Mg⁺² on Pichia stipitis growth and ethanol production was studied under condition of constant oxygen uptake rate (OUR) . Biomass/xylose and biomass/Mg⁺² yields increased with Mg⁺² concentration with a maximum value at Mg⁺² 4mM, ethanol being the main product obtained. At low Mg⁺² levels (ImM) 49 % of carbon flux to ethanol was redirected to xylitol production, accomplished through NADH intracellular accumulation.     

Cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, Pichia stipitis

Rising crude oil prices and environmental concerns have renewed interest in renewable energy. Cellulosic ethanol promises to deliver a renewable fuel from non-food feedstocks. One technical challenge producing cellulosic ethanol economically is a robust organism to utilize the different sugars present in cellulosic biomass. Unlike starch where glucose is the only sugar present, cellulosic biomass has other sugars such as xylose and arabinose, usually called C5 sugars. This review examines the most promising naturally occurring C5 fermenting organism, Pichia stipitis. In this work, the properties that make P. stipitis unique from other organisms, its physiology and fermentation results on lignocellulosic substrates have been reviewed. P. stipitis can produce 41 g ethanol/l with a potential to cleanup some of the most concentrated toxins. These results coupled with the less stringent nutritional requirements, great resistance to contamination and its thick cell walls makes P. stipitis a viable organism for scale-up. However, P. stipitis has a slower sugar consumption rate compared to Saccharomyces cerevisiae and requires microaerophilic condition for ethanol production. Finally, future studies to enhance fermentation capabilities of this yeast have been discussed.     

Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis

The inhibitory effect of the main inhibitors (acetic acid, furfural and 5-hydroxymethylfurfural) formed during steam explosion of wheat straw was studied through ethanol fermentations of model substrates and hydrolysates from wheat straw by Pichia stipitis. Experimental results showed that an increase in acetic acid concentration led to a reduction in ethanol productivity and complete inhibition was observed at 3.5 g/L. Furfural produced a delay on sugar consumption rates with increasing concentration and HMF did not exert a significant effect. Fermentations of the whole slurry from steam exploded wheat straw were completely inhibited by a synergistic effect due to the presence of 1.5 g/L acetic acid, 0.15 g/L furfural and 0.05 g/L HMF together with solid fraction. When using only the solid fraction from steam explosion, hydrolysates presented 0.5 g/L of acetic acid, whose fermentations have submitted promising results, providing an ethanol yield of 0.45 g ethanol/g sugars and the final ethanol concentration reached was 12.2 g/L (10.9 g ethanol/100 g DM).     

Effect of pretreatment chemicals on xylose fermentation by Pichia stipitis

Pretreatment of biomass with dilute H₂SO₄ results in residual acid which is neutralized with alkalis such as Ca(OH)₂, NaOH and NH₄OH. The salt produced after neutralization has an effect on the fermentation of Pichia stipitis. Synthetic media of xylose (60 g total sugar/l) was fermented to ethanol in the presence and absence of the salts using P. stipitis CBS 6054. CaSO₄ enhanced growth and xylitol production, but produced the lowest ethanol concentration and yield after 140 h. Na₂SO₄ inhibited xylitol production, slightly enhanced growth towards the end of fermentation but had no significant effect on xylose consumption and ethanol concentration. (NH₄)₂SO₄ inhibited growth, had no effect on xylitol production, and enhanced xylose consumption and ethanol production.     

Real‐time monitoring prefrontal activities during online video game playing by functional near‐infrared spectroscopy

A growing body of literature has suggested that video game playing can induce functional and structural plasticity of the brain. The underlying mechanisms, however, remain poorly understood. In this study, functional near‐infrared spectroscopy (fNIRS) was used to record prefrontal activities in 24 experienced game players when they played a massively multiplayer online battle arena video game, League of Legends (LOL), under naturalistic conditions. It was observed that game onset was associated with significant activations in the ventrolateral prefrontal cortex (VLPFC) and concomitant deactivations in the dorsolateral prefrontal cortex (DLPFC) and frontal pole area (FPA). Game events, such as slaying an enemy and being slain by an enemy evoked region‐specific time‐locked hemodynamic/oxygenation responses in the prefrontal cortex (PFC). It was proposed that the VLPFC activities during LOL playing are likely responses to visuo‐motor task load of the game, while the DLPFC/FPA activities may be involved in the constant shifts of attentional states and allocation of cognitive resources required by game playing. The present study demonstrated that it is feasible to use fNIRS to monitor real‐time prefrontal activity during online video game playing.      

Influence of metal addition on ethanol production with <Emphasis Type="Italic">Pichia stipitis</Emphasis> ATCC 58784

Trace metals always act as cofactors or coenzymes in many cellular processes. Deficiency or excess of some metals will affect the fermentation of lignocellulosic hydrolysate. In order to make sure the deficient or excessive states of metals in culture medium, metal contents analysis was conducted in Pichia stipitis ATCC 58784 cells, synthetic medium, and diluted acid hydrolysate of rice straw. The results showed that Cu, Ni, and Co were deficient, and Al was a little excessive. So the influences of Cu²⁺, Al³⁺, Ni²⁺, and Co²⁺ additions on the growth and ethanol production of ATCC 58784 were further researched. Low concentration additions of Cu²⁺ and Al³⁺ (<0.24 mM and <0.23 mM, respectively) improved biomass growth of ATCC 58784 by 34 and 13%, respectively; however, higher concentrations decreased biomass growth. On the other hand, addition of Cu²⁺ (0.39 mM) did not affect volumetric ethanol production significantly (P = 0.05) and addition of Al³⁺ (0.38 mM) showed no influence on volumetric ethanol production (P = 0.68). Addition of 0.074 mM Co²⁺ inhibited biomass growth of ATCC 58784 by 13% and volumetric ethanol production by 10%. The biomass growth and volumetric ethanol production of ATCC 58784 was arrested by the addition of 0.33 mM of Ni²⁺ by 53 and 65%, respectively.     

Real‐time monitoring of high‐gravity corn mash fermentation using in situ raman spectroscopy

In situ Raman spectroscopy was employed for real‐time monitoring of simultaneous saccharification and fermentation (SSF) of corn mash by an industrial strain of Saccharomyces cerevisiae. An accurate univariate calibration model for ethanol was developed based on the very strong 883 cm^?1 C–C stretching band. Multivariate partial least squares (PLS) calibration models for total starch, dextrins, maltotriose, maltose, glucose, and ethanol were developed using data from eight batch fermentations and validated using predictions for a separate batch. The starch, ethanol, and dextrins models showed significant prediction improvement when the calibration data were divided into separate high‐ and low‐concentration sets. Collinearity between the ethanol and starch models was avoided by excluding regions containing strong ethanol peaks from the starch model and, conversely, excluding regions containing strong saccharide peaks from the ethanol model. The two‐set calibration models for starch (R² = 0.998, percent error = 2.5%) and ethanol (R² = 0.999, percent error = 2.1%) provide more accurate predictions than any previously published spectroscopic models. Glucose, maltose, and maltotriose are modeled to accuracy comparable to previous work on less complex fermentation processes. Our results demonstrate that Raman spectroscopy is capable of real time in situ monitoring of a complex industrial biomass fermentation. To our knowledge, this is the first PLS‐based chemometric modeling of corn mash fermentation under typical industrial conditions, and the first Raman‐based monitoring of a fermentation process with glucose, oligosaccharides and polysaccharides present. Biotechnol. Bioeng. 2013; 110: 1654–1662. © 2013 Wiley Periodicals, Inc.     

Real‐time model based process monitoring of enzymatic biodiesel production

In this contribution we extend our modelling work on the enzymatic production of biodiesel where we demonstrate the application of a Continuous‐Discrete Extended Kalman Filter (a state estimator). The state estimator is used to correct for mismatch between the process data and the process model for Fed‐batch production of biodiesel. For the three process runs investigated, using a single tuning parameter, q_x = 2 × 10^?2 which represents the uncertainty in the process model, it was possible over the entire course of the reaction to reduce the overall mean and standard deviation of the error between the model and the process data for all of the five measured components (triglycerides, diglycerides, monoglycerides, fatty acid methyl esters, and free fatty acid). The most significant reduction for the three process runs, were for the monoglyceride and free fatty acid concentration. For those components, there was over a ten‐fold decrease in the overall mean error for the state estimator prediction compared with the predictions from the pure model simulations. It is also shown that the state estimator can be used as a tool for detection of outliers in the measurement data. For the enzymatic biodiesel process, given the infrequent and sometimes uncertain measurements obtained we see the use of the Continuous‐Discrete Extended Kalman Filter as a viable tool for real time process monitoring. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:585–595, 2015     

On-line monitoring of the transesterification reaction between triglycerides and ethanol using near infrared spectroscopy combined with gas chromatography

Many analytical procedures have been developed to determine the composition of reaction mixtures during transesterification of vegetable oils with alcohols. However, despite their accuracy, these methods are time consuming and cannot be easily used for on-line monitoring. In this work, a fast analytical method was developed to on-line monitor the transesterification reaction of high oleic sunflower oil with ethanol using Near InfraRed spectroscopy and a multivariate approach. The reactions were monitored through sequential scans of the reaction medium with a probe in a one-liter batch reactor without collecting and preparing samples. To calibrate the NIR analytical method, gas chromatography-flame ionization detection was used as a reference method. The method was validated by studying the kinetics of the EtONa-catalyzed transesterification reaction. Activation energy (51.0 kJ/mol) was also determined by considering a pseudo second order kinetics model.     

Comparison of different pretreatments in ethanol fermentation using corn cob hemicellulosic hydrolysate with Pichia stipitis and Candida shehatae

A hemicellulosic hydrolysate was prepared with 0.3 M H₂SO₄ at 98 °C for 1 h. The total initial reducing sugar was maintained at 45 g l^–1 by synthetic xylose supplementation. The seven detoxification methods were employed including either the single addition of solid CaO (to pH 10 or 6) or its combinations with zeolite shaking. Over-liming gave the hydrolysate that was most completely fermented by Pichia stipitis and Candida shehatae at 30 °C, pH 6, among the tested methods.     

Real‐time monitoring of incision profile during laser surgery using shock wave detection

Lack of sensory feedback during laser surgery prevents surgeons from discerning the exact location of the incision, which increases duration and complexity of the treatment. In this study we demonstrate a new method for monitoring of laser ablation procedures. Real‐time tracking of the exact three dimensional (3D) lesion profile is accomplished by detection of shock waves emanating from the ablation spot and subsequent reconstruction of the incision location using time‐of‐flight data obtained from multiple acoustic detectors. Here, incisions of up to 9 mm in depth, created by pulsed laser ablation of fresh bovine tissue samples, were successfully monitored in real time. It was further observed that, by utilizing as little as 12 detection elements, the incision profile can be characterized with accuracy below 0.5 mm in all three dimensions and in good agreement with histological examinations. The proposed method holds therefore promise for delivering high precision real‐time feedback during laser surgeries. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Improving the monitoring of methanol concentration during high cell density fermentation of Pichia pastoris

The Pichia pastoris expression system is widely used for the production of recombinant proteins. A simple and efficient experimental set-up allowing on-line monitoring of the methanol concentration during the fermentation of P. pastoris based on the detection of the methanol vapor concentration in the exhaust air from fermenter by a tin dioxide (SnO2) semiconductor sensor is described. An experimental procedure to allow precise calibration of the system and to reduce methanol sensor's interferences (>95% reduction) are also presented and discussed. Accuracy and measurement error were estimated about 0.05 g x l(-1) and 6%, respectively. The efficient monitoring of methanol will help to advanced control of recombinant protein production and process optimization.     

Real‐time monitoring of influenza virus production kinetics in HEK293 cell cultures

There is an increased interest from the vaccine industry to use mammalian cell cultures for influenza vaccine manufacturing. Therefore, it became important to study the influenza infection mechanism, the viral–host interaction, and the replication kinetics from a bioprocessing stand point to maximize the influenza viral production yield in cell culture. In the present work, influenza replication kinetics was studied in HEK293 cells. Two infection conditions were evaluated, a low (0.01) and a high multiplicity of infection (1.0). Critical time points of the viral production cycle (infection, protein synthesis, viral assembly and budding, viral release, and host‐cell death) were identified in small‐scale cell cultures. Additionally, cell growth, viability, and viral titers were monitored in the viral production process. The infection state of the cultivated cell population was assessed by influenza immunolabeling throughout the culture period. Influenza virus production kinetics were also on‐line monitored by dielectric spectroscopy and successfully correlated to real‐time capacitance measures. Overall, this work provided insights into the mechanisms associated with the infection of human HEK293 cell line by the influenza virus and demonstrated, once again, the usefulness of multifrequency scanning permittivity for in‐line monitoring and supervision of cell‐based viral production processes. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Production of ethanol from corn stover hemicellulose hydrolyzate using <Emphasis Type="Italic">Pichia stipitis</Emphasis>

Hemicellulose liquid hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol using Pichia stipitis CBS 6054. The fermentation rate increased with aeration but the pH also increased due to consumption of acetic acid by Pichia stipitis. Hemicellulose hydrolyzate containing 34 g/L xylose, 8 g/L glucose, 8 g/L Acetic acid, 0.73 g/L furfural, and 1 g/L hydroxymethyl furfural was fermented to 15 g/L ethanol in 72 h. The yield in all the hemicellulose hydrolyzates was 0.37–0.44 g ethanol/g (glucose + xylose). Nondetoxified hemicellulose hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol with high yields, and this has the potential to improve the economics of the biomass to ethanol process.     

Real‐time monitoring of antibody glycosylation site occupancy by in situ Raman spectroscopy during bioreactor CHO cell cultures

The glycosylation of therapeutic monoclonal antibodies (mAbs), a known critical quality attribute, is often greatly modified during the production process by animal cells. It is essential for biopharmaceutical industries to monitor and control this glycosylation. However, current glycosylation characterization techniques involve time‐ and labor‐intensive analyses, often carried out at the end of the culture when the product is already synthesized. This study proposes a novel methodology for real‐time monitoring of antibody glycosylation site occupancy using Raman spectroscopy. It was first observed in CHO cell batch culture that when low nutrient concentrations were reached, a decrease in mAb glycosylation was induced, which made it essential to rapidly detect this loss of product quality. By combining in situ Raman spectroscopy with chemometric tools, efficient prediction models were then developed for both glycosylated and nonglycosylated mAbs. By comparing variable importance in projection profiles of the prediction models, it was confirmed that Raman spectroscopy is a powerful method to distinguish extremely similar molecules, despite the high complexity of the culture medium. Finally, the Raman prediction models were used to monitor batch and feed‐harvest cultures in situ. For the first time, it was demonstrated that the concentrations of glycosylated and nonglycosylated mAbs could be successfully and simultaneously estimated in real time with high accuracy, including their sudden variations due to medium exchanges. Raman spectroscopy can thus be considered as a promising PAT tool for feedback process control dedicated to on‐line optimization of mAb quality. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:486–493, 2018     

基于近红外光谱实时监测乙醇发酵过程多组分参数

生物量、葡萄糖浓度和乙醇浓度是乙醇发酵过程的重要参数,传统的方法通常对发酵液取样作离线测量,不仅需要采用多种仪器进行测试分析,而且耗时耗力,成为实时过程调控和优化的障碍。文中针对这些重要过程参数提出了一个基于近红外光谱技术的原位实时检测方法。通过采用浸入式近红外光谱仪对发酵溶液进行原位测量,基于多输出最小二乘支持向量机回归(MLS-SVR)方法建立了利用近红外光谱同时分析葡萄糖浓度、生物量和乙醇浓度的多输出预测模型。实验结果表明,该方法能实时准确地检测乙醇发酵过程中的葡萄糖浓度、生物量和乙醇浓度,而且相对于现有的偏最小二乘法(PLS)分别对各组分建模和预测,能明显提高测量准确性和可靠性。     

Advanced near‐infrared monitor for stable real‐time measurement and control of Pichia pastoris bioprocesses

Near‐infrared spectroscopy is considered to be one of the most promising spectroscopic techniques for upstream bioprocess monitoring and control. Traditionally the nature of near‐infrared spectroscopy has demanded multivariate calibration models to relate spectral variance to analyte concentrations. The resulting analytical measurements have proven unreliable for the measurement of metabolic substrates for bioprocess batches performed outside the calibration process. This paper presents results of an innovative near‐infrared spectroscopic monitor designed to follow the concentrations of glycerol and methanol, as well as biomass, in real time and continuously during the production of a monoclonal antibody by a Pichia pastoris high cell density process. A solid state instrumental design overcomes the ruggedness limitations of conventional interferometer‐based spectrometers. Accurate monitoring of glycerol, methanol, and biomass is demonstrated over 274 days postcalibration. In addition, the first example of feedback control to maintain constant methanol concentrations, as low as 1 g/L, is presented. Postcalibration measurements over a 9‐month period illustrate a level of reliability and robustness that promises its adoption for online bioprocess monitoring throughout product development, from early laboratory research and development to pilot and manufacturing scale operation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:749–759, 2014