Raman spectroscopy is a suitable monitoring technique for CHO cultivations. However, a thorough discussion of peaks, bands, and region assignments to key metabolites and culture attributes, and the interpretability of produced calibrations is scarce. That understanding is vital for the long-term predictive ability of monitoring models, and to facilitate lifecycle management that comply with regulatory guidelines. Several fed-batch lab-scale mAb mammalian cultivations were carried out, with in situ Raman spectroscopy used for process state estimation and attribute monitoring. The goal was to evaluate its use as a process analytical technology (PAT) tool to detect residual glucose and lactate levels, understand their dynamics and interconversion, and eventually estimate key performance culture and product quality attributes. Glucose and lactate models were optimized up to 0.31 g L with 3 Latent Variables (LVs) and 0.19 g L (2 LVs) accuracy, respectively. Glutamine and product titer models, were not specific and accurate enough, even though indirect calibrations were obtained with a RMSEP of 0.12 g L (4 LVs) and 0.29 g L (5 LVs), respectively. A critical discussion and details about the extensive work done in calibration development and optimization are provided. Namely, considering a risk-based selection of variability sources impacting sample spectra, executing designed experiments with spiked cultivations, and using advanced chemometric procedures for variable selection and model cross validation. A strategy is presented to evaluation Raman spectroscopy as a reliable PAT technology fit-for industrial use. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:659-670, 2018.

The metabolic and energetic characterization of the growth of Leuconostoc oenos on glucose-citrate or glucose-fructose mixtures enables the potential role of this bacterium in the wine-making process to be ascertained. Moreover, mixotrophic conditions remain a suitable means for improving biomass productivities of malolactic starter cultures. When the malolactic bacterium L. oenos was grown in batch cultures on complex medium at pH 5.0 with glucose-citrate or glucose-fructose mixtures, enhancement of both the specific growth rate and biomass production yields was observed. While growth was possible on fructose as the sole source of energy, citrate alone did not allow subsequent biomass production. The metabolic interactions between the catabolic pathways of the glucose cosubstrates and the heterofermentation of hexoses led to an increased acetate yield as a result of modified NADH oxidation. However, the calculated global coenzyme regeneration showed that the reducing equivalent balance was never equilibrated. The stimulatory efects of these glucose cosubstrates on growth resulted from increased ATP synthesis by substrate-level phosphorylation via acetate kinase. While the energetic efficiency remained close to 10 g of biomass produced per mol of ATP, the increase in the specific growth rate and biomass production yields was directly related to the rate and yield of ATP generation.Leuconostoc oenos has the ability to convert L-( -)-malate to L-(+)-lactate and carbon dioxide in a single step catalyzed by L-malate:NAD' carboxylyase. This so-called malolactic fermentation is an important reaction in the vinification process, since it reduces acidity, enhances organoleptic characteristics, and improves the microbiological stability of wines (3). For several years, wine makers have tended to use selected malolactic bacteria to ensure better control of malic acid bioconversion in enology. Thus, studies have been performed to examine the use of L. oenos strains as malolactic starter cultures, with the aims of both optimizing growth and understanding better the metabolism of this bacterium.Glucose remains the most frequently employed catabolic substrate providing the energy necessary for the growth of Leuconostoc species. Hexoses are metabolized via the heterofermentative phosphoketolase pathway or the 6-phosphogluconate pathway, leading to equimolar amounts of lactate, C02, and ethanol-acetate (6). On the other hand, it is well known that a number of lactic acid bacteria, including Leuconostoc species, are able to metabolize citrate. Since citrate catabolism is of importance for the production of flavor compounds, such as diacetyl and acetoin, numerous studies have dealt with the cometabolism of citrate and sugars. However, many of these studies are difficult to interpret because the relationship between the substrates used and the products formed was not quantified. Recently, Cogan (2) described the kinetics of substrate utilization and product formation from glucose and glucose-citrate mixtures during the growth of se...

Growth of the malolactic bacterium Leuconostoc oenos was improved with respect to both the specific growth rate and the biomass yield during the fermentation ofglucose-malate mixtures as compared with those in media lacking malate. Such a finding indicates that the malolactic reaction contributed to the energy budget of the bacterium, suggesting that growth is energy limited in the absence of malate. An energetic yield (YATP) of 9.5 g of biomass mol ATP'1 was found during growth on glucose with an ATP production by substrate-level phosphorylation of 1.2 mol of ATP. mol of glucose-'. During the period of mixed-substrate catabolism, an apparent YATP of 17.7 was observed, indicating a mixotrophy-associated ATP production of 2.2 mol of ATP-mol of glucose 1, or more correctly an energy gain of 0.28 mol of ATP-mol of malate-1, representing proton translocation flux from the cytoplasm to the exterior of 0.56 or 0.84 H+ mol of malate'1 (depending on the H+/ATP stoichiometry). The growth-stimulating effect of malate was attributed to chemiosmotic transport mechanisms rather than proton consumption by the malolactic enzyme. Lactate efflux was by electroneutral lactatef/H+ symport having a constant stoichiometry, while malate uptake was predominantly by a malate-/H+ symport, though a low-affinity malate-uniport was also implicated. The measured electrical component (A*) of the proton motive force was altered, passing from -30 to -60 mV because of this translocation of dissociated organic acids when malolactic fermentation occurred.Certain species of lactic acid bacteria (belonging to the genera Lactobacillus, Pediococcus, and Leuconostoc) possess the capacity to convert malate to lactate via a direct decarboxylation reaction referred to as malolactic fermentation. This fermentation is of use in the enological industry since it enables the removal of excess acidity, enhances organoleptic properties, and increases the bacteriological stability of wine (7,33).Those bacteria able to metabolize malate via malolactic fermentation show improved growth characteristics when presented with glucose-malate mixtures as compared with the growth characteristics during fermentation of glucose alone. This response to an auxiliary substrate is not, at first glance, surprising: increased growth yields attributable to a second catabolic substrate have been previously reported (2, 13). In these reports the maximum specific growth rates remained unaltered, being fixed by carbon flux limitations. More recently, improved growth yields and specific growth rates have been achieved with substrate mixtures which overcome both carbon flux and energetic limitations simultaneously (15). In the case of the malolactic bacteria, the situation is somewhat different in that the malate cannot contribute directly to the anabolic carbon flux and, furthermore, there is no energy conservation at the enzyme level (either directly by substrate-level phosphorylation [SLP] or indirectly via reducing equivalent generation). The reaction involved in the decarboxylatio...

Growth, substrate utilization and product formation were studied in batch cultures of a Leuconostoc oenos strain. The effect of various culture conditions, i.e. pH-control at different values and various initial concentrations of malate and glucose, on growth and metabolism were investigated. Addition of malate resulted in a marked stimulation of growth, with only a slight increase in final biomass but a high conversion yield of glucose. Under pH control this stimulation was much greater than could be accounted for from changes in pH profile resulting from malate utilization. The specific rate of malate utilization was maximal at pH 4.0 whereas the specific rate of glucose consumption was highest at pH 5.5. During co-metabolism of malic acid and glucose, substrate utilization and product formation agreed with the stoichiometric relationships of the malo-lactic reaction and the heterolactic fermentation of glucose.