Cell culture process metabolomics together with multivariate data analysis tools opens new routes for bioprocess development and glycosylation prediction.

Multivariate latent variable methods have become a popular and versatile toolset to analyze bioprocess data in industry and academia. This work spans such applications from the evaluation of the role of the standard process variables and metabolites to the metabolomics level, that is, to the extensive number metabolic compounds detectable in the extracellular and intracellular domains. Given the substantial effort currently required for the measurement of the latter groups, a tailored methodology is presented that is capable of providing valuable process insights as well as predicting the glycosylation profile based on only four experiments measured over 12 cell culture days. An important result of the work is the possibility to accurately predict many of the glycan variables based on the information of three experiments. An additional finding is that such predictive models can be generated from the more accessible process and extracellular information only, that is, without including the more experimentally cumbersome intracellular data. With regards to the incorporation of omics data in the standard process analytics framework in the future, this works provides a comprehensive data analysis pathway which can efficiently support numerous bioprocessing tasks.

Outward electron transfer by Saccharomyces cerevisiae monitored with a bi-cathodic microbial fuel cell-type activity sensor.

A Janus head-like bi-cathodic microbial fuel cell was constructed to monitor the electron transfer from Saccharomyces cerevisiae to a woven carbon anode. The experiments were conducted during an ethanol cultivation of 170 g/l glucose in the presence and absence of yeast-peptone medium. First, using a basic fuel-cell type activity sensor, it was shown that yeast-peptone medium contains electroactive compounds. For this purpose, 1% solutions of soy peptone and yeast extract were subjected to oxidative conditions, using a microbial fuel cell set-up corresponding to a typical galvanic cell, consisting of culture medium in the anodic half-cell and 0.5 M K(3)Fe(CN)(6) in the cathodic half-cell. Second, using a bi-cathodic microbial fuel cell, it was shown that electrons were transferred from yeast cells to the carbon anode. The participation of electroactive compounds in the electron transport was separated as background current. This result was verified by applying medium-free conditions, where only glucose was fed, confirming that electrons are transferred from yeast cells to the woven carbon anode. Knowledge about the electron transfer through the cell membrane is of importance in amperometric online monitoring of yeast fermentations and for electricity production with microbial fuel cells.

Online monitoring of yeast cultivation using a fuel-cell-type activity sensor.

A microbial fuel-cell type activity sensor integrated into 500 mL and 3.2 L bioreactors was employed for ampero- (lA) and potentiometric (mV) measurements. The aim was to follow the microbial activity during ethanol production by Saccharomyces cerevisiae and to detect the end of carbohydrate consumption. Three different sensor setups were tested to record electrochemical signals produced by the metabolism of glucose and fructose (1:1) online. In a first setup, a reference electrode was used to record the potentiometric values, which rose from 0.26 to 0.5 V in about 10 h during the growth phase. In a second setup, a combination of ampero- and pseudo-potentiometric measurements delivered a maximum voltage of 35 mV. In this arrangement, the pseudo-potentiometric signal changed in a manner that was directly proportional to the amperometric signals, which reached a maximum value of 32 muA. In a third type of arrangement, a reference electrode was added to the anodic bioreactor compartment to carry out ampero- and potentiometric measurements; this is made possible by the high internal resistance of the cultivation. In this case, the reference potential rose to 0.44 V while the current maximum recorded by the working electrodes reached 27 lA. Reference and pseudo-reference electrodes were in all cases K(3)Fe(CN)(6)/carbon. Electrodes were made of 9 cm(2) woven graphite. To compare the electrochemical signals with established values, the metabolism was also monitored for optical density (at 600 nm) indicating biomass production. For fructose and glucose conversion, HPLC with an Aminex column and RI detector was used, and ethanol production was analyzed by GC with methanol as internal standard. The combination of amperometric and potentiometric recordings was found to be an ideal setup and was successfully used in reproducible cultivations.