Quantitative Flow Cytometry to Understand Population Heterogeneity in Response to Changes in Substrate Availability in Escherichia coli and Saccharomyces cerevisiae Chemostats.

Microbial cells in bioprocesses are usually described with averaged parameters. But in fact, single cells within populations vary greatly in characteristics such as stress resistance, especially in response to carbon source gradients. Our aim was to introduce tools to quantify population heterogeneity in bioprocesses using a combination of reporter strains, flow cytometry, and easily comprehensible parameters. We calculated mean, mode, peak width, and coefficient of variance to describe distribution characteristics and temporal shifts in fluorescence intensity. The skewness and the slope of cumulative distribution function plots illustrated differences in distribution shape. These parameters are person-independent and precise. We demonstrated this by quantifying growth-related population heterogeneity of Saccharomyces cerevisiae and Escherichia coli reporter strains in steady-state of aerobic glucose-limited chemostat cultures at different dilution rates and in response to glucose pulses. Generally, slow-growing cells showed stronger responses to glucose excess than fast-growing cells. Cell robustness, measured as membrane integrity after exposure to freeze-thaw treatment, of fast-growing cells was strongly affected in subpopulations of low membrane robustness. Glucose pulses protected subpopulations of fast-growing but not slower-growing yeast cells against membrane damage. Our parameters could successfully describe population heterogeneity, thereby revealing physiological characteristics that might have been overlooked during traditional averaged analysis.

An integrated strategy for the effective production of bristle protein hydrolysate by the keratinolytic filamentous bacterium Amycolatopsis keratiniphila D2.

In a conventional microorganism-mediated biological process for degradation of keratinous waste material the production of keratin-specific proteases (i.e., keratinases) and the hydrolysis of keratin-rich residual biomass both take place during the same stage of the bioprocess and, as a consequence, occur simultaneously under suboptimal conditions. In the present study the keratinolytic actinomycete Amycolatopsis keratiniphila D2 was successfully employed to biodegrade thermally pretreated porcine bristles at high solids loading (16% w/v) via a novel cultivation methodology. Indeed, the two-stage submerged fermentation process developed in this work enabled to efficiently recover, in a single unit operation, about 73% of the protein material contained in the keratinous biowaste structure, resulting in an overall accumulation of 89.3 g·L-1 protein-rich hydrolysate and a productivity of 427 mg crude soluble proteins per litre per hour. The obtained protein hydrolysate powder displayed a 2.2-fold increase in its in vitro pepsin digestibility (95%) with respect to the non-hydrolysed pretreated substrate (43%). In addition, the chromatogram obtained by size-exclusion chromatography analysis of the final product indicated that, among the identified fractions, those consisting of small peptides and free amino acids were the most abundantly present inside the analysed sample. Given these facts it is possible to conclude that the soluble proteins, peptides and free amino acids recovered through the newly designed two-stage bioextraction process could represent a viable alternative source of protein in animal feed formulation.

Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae.

A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by placing it in strains with different ability to reoxidise NADH, and applying different environmental conditions. Flow cytometric analysis of reporter strains expressing green fluorescent protein (GFP) under the control of the GPD2 promoter was used to determine the promoter activity at the single-cell level. When placed in a gpd1Δgpd2Δ strain background, the GPD2 promoter displayed a 2-fold higher activity as compared to the strong constitutive glyceraldehyde-3-phosphate dehydrogenase (TDH3). In contrast, the GPD2 promoter was found to be inactive when cells were cultivated in continuous mode at a growth rate of 0.3 h-1 and in conditions with excess oxygen (i.e. with an aeration of 2.5 vvm, and a stirring of 800 rpm). In addition, a clear window of operation where the gpd1Δgpd2Δ strain can be grown with the same efficiency as wild type yeast was identified. In conclusion, the flow cytometry mapping revealed conditions where the GPD2 promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression.

Applying mechanistic models in bioprocess development.

The available knowledge on the mechanisms of a bioprocess system is central to process analytical technology. In this respect, mechanistic modeling has gained renewed attention, since a mechanistic model can provide an excellent summary of available process knowledge. Such a model therefore incorporates process-relevant input (critical process variables)-output (product concentration and product quality attributes) relations. The model therefore has great value in planning experiments, or in determining which critical process variables need to be monitored and controlled tightly. Mechanistic models should be combined with proper model analysis tools, such as uncertainty and sensitivity analysis. When assuming distributed inputs, the resulting uncertainty in the model outputs can be decomposed using sensitivity analysis to determine which input parameters are responsible for the major part of the output uncertainty. Such information can be used as guidance for experimental work; i.e., only parameters with a significant influence on model outputs need to be determined experimentally. The use of mechanistic models and model analysis tools is demonstrated in this chapter. As a practical case study, experimental data from Saccharomyces cerevisiae fermentations are used. The data are described with the well-known model of Sonnleitner and Käppeli (Biotechnol Bioeng 28:927-937, 1986) and the model is analyzed further. The methods used are generic, and can be transferred easily to other, more complex case studies as well.

Introducing process analytical technology (PAT) in filamentous cultivation process development: comparison of advanced online sensors for biomass measurement.

The recent process analytical technology (PAT) initiative has put an increased focus on online sensors to generate process-relevant information in real time. Specifically for fermentation, however, introduction of online sensors is often far from straightforward, and online measurement of biomass is one of the best examples. The purpose of this study was therefore to compare the performance of various online biomass sensors, and secondly to demonstrate their use in early development of a filamentous cultivation process. Eight Streptomyces coelicolor fed-batch cultivations were run as part of process development in which the pH, the feeding strategy, and the medium composition were varied. The cultivations were monitored in situ using multi-wavelength fluorescence (MWF) spectroscopy, scanning dielectric (DE) spectroscopy, and turbidity measurements. In addition, we logged all of the classical cultivation data, such as the carbon dioxide evolution rate (CER) and the concentration of dissolved oxygen. Prediction models for the biomass concentrations were estimated on the basis of the individual sensors and on combinations of the sensors. The results showed that the more advanced sensors based on MWF and scanning DE spectroscopy did not offer any advantages over the simpler sensors based on dual frequency DE spectroscopy, turbidity, and CER measurements for prediction of biomass concentration. By combining CER, DE spectroscopy, and turbidity measurements, the prediction error was reduced to 1.5 g/l, corresponding to 6% of the covered biomass range. Moreover, by using multiple sensors it was possible to check the quality of the individual predictions and switch between the sensors in real time.

Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations.

BACKGROUND: Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted. RESULTS: Two minimal defined media, one with low Pi (0.6 mM; LP) and proficient glucose (12 g/l) concentrations and the other one with high Pi (1.8 mM) and limiting (6 g/l; LG) glucose concentrations, were developed to promote and repress antibiotic production, respectively, in A. balhimycina chemostat cultivations. Applying the same dilution rate (0.03 h-1), both LG and LP chemostat cultivations showed a stable steady-state where biomass production yield coefficients, calculated on glucose consumption, were 0.38 ± 0.02 and 0.33 ± 0.02 g/g (biomass dry weight/glucose), respectively. Notably, balhimycin was detected only in LP, where quantitative RT-PCR revealed upregulation of selected bal genes, devoted to balhimycin biosynthesis, and of phoP, phoR, pstS and phoD, known to be associated to Pi limitation stress response. 2D-Differential Gel Electrophoresis (DIGE) and protein identification, performed by mass spectrometry and computer-assisted 2 D reference-map http://www.unipa.it/ampuglia/Abal-proteome-maps matching, demonstrated a differential expression for proteins involved in many metabolic pathways or cellular processes, including central carbon and phosphate metabolism. Interestingly, proteins playing a key role in generation of primary metabolism intermediates and cofactors required for balhimycin biosynthesis were upregulated in LP. Finally, a bioinformatic approach showed PHO box-like regulatory elements in the upstream regions of nine differentially expressed genes, among which two were tested by electrophoresis mobility shift assays (EMSA). CONCLUSION: In the two chemostat conditions, used to generate biomass for proteomic analysis, mycelia grew with the same rate and with similar glucose-biomass conversion efficiencies. Global gene expression analysis revealed a differential metabolic adaptation, highlighting strategies for energetic supply and biosynthesis of metabolic intermediates required for biomass production and, in LP, for balhimycin biosynthesis. These data, confirming a relationship between primary metabolism and antibiotic production, could be used to increase antibiotic yield both by rational genetic engineering and fermentation processes improvement.

Application of microbioreactors in fermentation process development: a review.

Biotechnology process development involves strain testing and improvement steps aimed at increasing yields and productivity. This necessitates the high-throughput screening of many potential strain candidates, a task currently mainly performed in shake flasks or microtiter plates. However, these methods have some drawbacks, such as the low data density (usually only end-point measurements) and the lack of control over cultivation conditions in standard shake flasks. Microbioreactors can offer the flexibility and controllability of bench-scale reactors and thus deliver results that are more comparable to large-scale fermentations, but with the additional advantages of small size, availability of online cultivation data and the potential for automation. Current microbioreactor technology is analyzed in this review paper, focusing on its industrial applicability, and directions for future research are presented.

Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation.

Industrial enzymes are often produced by filamentous fungi in fed-batch cultivations. During cultivation, the different morphological forms displayed by the fungi have an impact on the overall production. The morphology of a recombinant lipase producing Aspergillus oryzae strain was investigated during fed-batch cultivations. During the exponential batch phase of the fed-batch cultivations, the average hyphal length increased as did the number of tips per hyphal element. Most striking was the finding that the diameter of the hyphal elements increased with an average factor of 1.5 during the batch phase from 2.8-2.9 up to 4.0-4.4 mum. The diameter of the hyphal elements remained constant, around 4 mum, after the feed was started. However, the diameter of the immediate hyphal tip, where the enzyme secretion is thought to take place, increased dramatically with up to a factor 2.5 during the feeding period. The expression of the recombinant lipase was induced by the feeding with maltose, and an increase in lipase activity was seen in parallel to a swelling of the tips. The results indicate that the two events are linked as a return to normal growth was observed upon cessation of production due to oxygen limitations.

Determination of cell mass and polymyxin using multi-wavelength fluorescence.

Multi-wavelength fluorescence was applied for on-line monitoring of cell mass and the antibiotic polymyxin B in Bacillus polymyxa cultivations. By varying the phosphate and nitrogen content of the medium different polymyxin-cell mass ratios could be obtained. Using this strategy, it was possible to investigate if multi-wavelength fluorescence is able to give independent prediction of the two parameters. Partial least square (PLS) regression was applied to establish mathematical relationships between off-line determined cell mass and polymyxin concentrations and on-line collected fluorescence data. For polymyxin one universal PLS model, with a correlation of 0.95 and a root mean square error of cross validation (RMSECV) of 35 mgl(-1), could be constructed. However, correlation between fluorescence and cell mass dry weight could not be established including data from all three types of cultivations. For data from each type of cultivation, separate models with high correlation and low RMSECV values were built. A large variation in cellular composition as a result of the different levels of nitrogen and phosphorus in the cultivations was the probable reason to the necessity of building three models. The results of the present investigation indicate that production of polymyxin is concomitantly regulated by phosphate and nitrogen as the highest polymyxin yield on cell mass, 0.17+/-0.01 gg(-1), was reached in the cultivations where both nitrogen and phosphate concentrations were kept low.