Switching the mode of sucrose utilization by Saccharomyces cerevisiae

Abstract Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae.

Developing a scalable model of recombinant protein yield from Pichia pastoris:the influence of culture conditions, biomass and induction regime

Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time.

Characterisation of a harvesting step for monoclonal antibodies from mammalian cell culture

The focus of this research was defined by a poorly characterised filtration train employed to clarify culture broth containing monoclonal antibodies secreted by GS-NSO cells: the filtration train blinded unpredictably and the ability of the positively charged filters to adsorb DNA from process material was unknown. To direct the development of an assay to quantify the ability of depth filters to adsorb DNA, the molecular weight of DNA from a large-scale, fed-batch, mammalian cell culture vessel was evaluated as process material passed through the initial stages of the purification scheme. High molecular weight DNA was substantially cleared from the broth after passage through a disc stack centrifuge and the remaining low molecular weight DNA was largely unaffected by passage through a series of depth filters and a sterilising grade membrane. Removal of high molecular weight DNA was shown to be coupled with clarification of the process stream. The DNA from cell culture supernatant showed a pattern of internucleosomal cleavage of chromatin when fractionated by electrophoresis but the presence of both necrotic and apoptotic cells throughout the fermentation meant that the origin of the fragmented DNA could not be unequivocally determined. An intercalating fluorochrome, PicoGreen, was elected for development of a suitable DNA assay because of its ability to respond to low molecular weight DNA. It was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing pertinent monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 89.0 % of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples. Application of the fluorescence based assay resulted in characterisation of the physical parameters governing adsorption of DNA by various positively charged depth filters and membranes in test solutions and the DNA adsorption profile of the manufacturing scale filtration train. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. Production-scale centrifugation of harvest broth containing therapeutic protein resulted in the reduction of total DNA in the process stream from 79.8 μg m1-1 to 9.3 μg m1-1 whereas the concentration of DNA in the supernatant of pre-and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 μg m1-1 respectively. Hence the filtration train was shown to ineffective in DNA removal. Historically, blinding of the depth filters had been unpredictable with data such as numbers of viable cells, non-viable cells, product titre, or process shape (batch, fed-batch, or draw and fill) failing to inform on the durability of depth filters in the harvest step. To investigate this, key fouling contaminants were identified by challenging depth filters with the same mass of one of the following: viable healthy cells, cells that had died by the process of apoptosis, and cells that had died through the process of necrosis. The pressure increase across a Cuno Zeta Plus 10SP depth filter was 2.8 and 16.5 times more sensitive to debris from apoptotic and necrotic cells respectively, when compared to viable cells. The condition of DNA released into the culture broth was assessed. Necrotic cells released predominantly high molecular weight DNA in contrast to apoptotic cells which released chiefly low molecular weight DNA. The blinding of the filters was found to be largely unaffected by variations in the particle size distribution of material in, and viscosity of, solutions with which they were challenged. The exceptional response of the depth filters to necrotic cells may suggest the cause of previously noted unpredictable filter blinding whereby a number of necrotic cells have a more significant impact on the life of a depth filter than a similar number of viable or apoptotic cells. In a final set of experiments the pressure drop caused by non-viable necrotic culture broths which had been treated with DNase I or benzonase was found to be smaller when compared to untreated broths: the abilities of the enzyme treated cultures to foul the depth filter were reduced by 70.4% and 75.4% respectively indicating the importance of DNA in the blinding of the depth filter studied.

Nitrogen removal in a sequencing batch biofilm reactor with liquid circulation: Effect of feed strategy and carbon source in the denitrifying step

Ammonium nitrogen removal from a synthetic wastewater by nitrification and denitrification processes were performed in a sequencing batch biofilm reactor containing immobilized biomass on polyurethane foam with circulation of the liquid-phase. It was analyzed the effect of four external carbon sources (ethanol, acetate, carbon synthetic medium and methanol) acting as electron donors in the denitrifying process. The experiments were conducted with intermittent aeration and operated at 30+/-1 degrees C in 8-h cycles. The synthetic wastewater (100 mgCOD/L and 50 mgNH(4)(+)-N/L) was added batch-wise, while the external carbon sources were added fed-batch-wise during the periods where aeration was suspended. Ammonium nitrogen removal efficiencies obtained were 95.7, 94.3 and 97.5% for ethanol, acetate and carbon synthetic medium, respectively. As to nitrite, nitrate and ammonium nitrogen effluent concentrations, the results obtained were, respectively: 0.1, 5.7 and 1.4 mg/L for ethanol; 0.2, 4.1 and 1.8 mg/L for acetate and 0.2, 6.7 and 0.8 for carbon synthetic medium. On the other hand using methanol, even at low concentrations (50% of the stoichiometric value calculated for complete denitrification), resulted in increasing accumulation of nitrate and ammonium nitrogen in the effluent over time.

Anaerobic sequencing batch biofilm reactor applied to automobile industry wastewater treatment: Volumetric loading rate and feed strategy effects

This paper presents a technological viability study of wastewater treatment in an automobile industry by an anaerobic sequencing batch biofilm reactor containing immobilized biomass (AnSBBR) with a draft tube. The reactor was operated in 8-h cycles, with agitation of 400 rpm, at 30 degrees C and treating 2.0 L wastewater per cycle. Initially the efficiency and stability of the reactor were studied when supplied with nutrients and alkalinity. Removal efficiency of 88% was obtained at volumetric loading rate (VLR) of 3.09 mg COD/L day. When VLR was increased to 6.19 mg COD/L day the system presented stable operation with reduction in efficiency of 71%. In a second stage the AnSBBR was operated treating wastewater in natura, i.e., without nutrients supplementation, only with alkalinity, thereby changing feed strategy. The first strategy consisted in feeding 2.0 L batch wise (10 min), the second in feeding 1.0 L of influent batch wise (10 min) and an additional 1.0 L fed-batch wise (4 h), both dewatering 2.0 L of the effluent in 10 min. The third one maintained 1.0 L of treated effluent in the reactor, without discharging, and 1.0 L of influent was fed fed-batch wise (4 h) with dewatering 1.0 L of the effluent in 10 min. For all implemented strategies (VLR of 1.40, 2.57 and 2.61 mg COD/L day) the system presented stability and removal efficiency of approximately 80%. These results show that the AnSBBR presents operational flexibility, as the influent can be fed according to industry availability. In industrial processes this is a considerable advantage, as the influent may be prone to variations. Moreover, for all the investigated conditions the kinetic parameters were obtained from fitting a first-order model to the profiles of organic matter, total volatile acids and methane concentrations. Analysis of the kinetic parameters showed that the best strategy is feeding 1.0 L of influent batchwise (10 min) and 1.0 L fed-batch wise (4 h) in 8-h cycle. (c) 2007 Elsevier B.V. All rights reserved.

Improved Bioprocess with CHO-hTSH Cells on Higher Microcarrier Concentration Provides Higher Overall Biomass and Productivity for rhTSH

Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH) cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium every 24 h. Cell cultures were monitored for agitation (rpm), temperature (A degrees C), dissolved oxygen (% DO), pH, cell concentration, MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with a repeated fed batch mode of operation at 40 rpm, 37 A degrees C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 x 10(6) cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high percent (similar to 80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (similar to 9 x 10(5) cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce similar to 1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity.

Metabolic analysis of poly(3-hydroxybutyrate) production by recombinant Escherichia coli

Poly(3-hydroxybutyrate) (PHB) production by fermentation was examined under both restricted- and ample-oxygen supply conditions in a single fed-batch fermentation. Recombinant Escherichia coli transformed with the PHB production plasmid pSYL107 was grown to reach high cell density (227 g/l dry cell weight) with a high PHB content (78% of dry cell weight), using a glucose-based minimal medium. A simple flux model containing 12 fluxes was developed and applied to the fermentation data. A superior closure (95%) of the carbon mass balance was achieved. When the data were put into use, the results demonstrated a surprisingly large excretion of formate and lactate. Even though periods of severe oxygen limitation coincided with rapid acetate and lactate excretion, PHB productivity and carbon utilization efficiency were not significantly impaired. These results are very positive in reducing oxygen demand in an industrial PHA fermentation without sacrificing its PHA productivity, thereby reducing overall production costs.

Osmolarity effects on observed insect cell size after baculovirus infection are avoided using growth medium for sample dilution

Rates of cell size increase are an important measure of success during the baculovirus infection process. Batch and fed batch cultures sustain large fluctuations in osmolarity that can affect the measured cell volume if this parameter is not considered during the sizing protocol. Where osmolarity differences between the sizing diluent and the culture broth exist, biased measurements of size are obtained as a result of the cell osmometer response. Spodoptera frugiperda (Sf9) cells are highly sensitive to volume change when subjected to a change in osmolarity. Use of the modified protocol with culture supernatants for sample dilution prior to sizing removed the observed error during measurement.

Process development for a recombinant Chinese Hamster Ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system

The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 muM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 107 cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system. (C) 2004 Wiley Periodicals, Inc.

Cell cycle model to describe animal cell size variation and lag between cell number and biomass dynamics

The use of cell numbers rather than mass to quantify the size of the biotic phase in animal cell cultures causes several problems. First, the cell size varies with growth conditions, thus yields expressed in terms of cell numbers cannot be used in the normal mass balance sense. Second, experience from microbial systems shows that cell number dynamics lag behind biomass dynamics. This work demonstrates that this lag phenomenon also occurs in animal cell culture. Both the lag phenomenon and the variation in cell size are explained using a simple model of the cell cycle. The basis for the model is that onset of DNA synthesis requires accumulation of G1 cyclins to a prescribed level. This requirement is translated into a requirement for a cell to reach a critical size before commencement of DNA synthesis. A slower gl-owing cell will spend more time in G1 before reaching the critical mass. In contrast, the period between onset of DNA synthesis and mitosis, tau(B), is fixed. The two parameters in the model, the critical size and tau(B), were determined from eight steady-state measurements of mean cell size in a continuous hybridoma culture. Using these parameters, it was possible to predict with reasonable accuracy the transient behavior in a separate shift-up culture, i.e., a culture where cells were transferred from a lean environment to a rich environment. The implications for analyzing experimental data for animal cell culture are discussed. (C) 1997 John Wiley & Sons, Inc.

Transport mechanism in ionic liquid membranes and the study of thiomersal biodegradation

The initial goal of this work was the development of a supported liquid membrane (SLM) bioreactor for the remediation of vaccine production effluents contaminated with a highly toxic organomercurial – thiomersal. Therefore, two main aspects were focused on: 1) the development of a stable supported liquid membrane – using room temperature ionic liquids (RTILs) – for the selective transport of thiomersal from the wastewater to a biological compartment, 2) study of the biodegradation kinetics of thiomersal to metallic mercury by a Pseudomonas putida strain. The first part of the work focused on the evaluation of the physicochemical properties of ionic liquids and on the SLMs’ operational stability. The results obtained showed that, although it is possible to obtain a SLM with a high stability, water possesses nonnegligible solubility in the RTILs studied. The formation of water clusters inside the hydrophobic ionic liquid was identified and found to regulate the transport of water and small ions. In practical terms, this meant that, although it was possible to transport thiomersal from the vaccine effluent to the biological compartment, complete isolation of the microbial culture could not be guaranteed and the membrane might ultimately be permeable to other species present in the aqueous vaccine wastewater. It was therefore decided not to operate the initially targeted integrated system but, instead, the biological system by itself. Additionally, attention was given to the development of a thorough understanding of the transport mechanisms involved in the solubilisation and transport of water through supported liquid membranes with RTILs as well as to the evaluation of the effect of water uptake by the SLM in the transport mechanisms of water-soluble solutes and its effect on SLM performance. The results obtained highlighted the determinant role played by water – solubilised inside the ionic liquids – on the transport mechanism. It became clear that the transport mechanism of water and water-soluble solutes through SLMs with [CnMIM][PF6] RTILs was regulated by the dynamics of water clusters inside the RTIL, rather than by molecular diffusion through the bulk of the ionic liquid. Although the stability tests vi performed showed that there were no significant losses of organic phase from the membrane pores, the formation of water clusters inside the ionic liquid, which constitute new, non-selective environments for solute transport, leads to a clear deterioration of SLM performance and selectivity. Nevertheless, electrical impedance spectroscopy characterisation of the SLMs showed that the formation of water clusters did not seem to have a detrimental effect on the SLMs’ electrical characteristics and highlighted the potential of using this type of membranes in electrochemical applications with low resistance requirements. The second part of the work studied the kinetics of thiomersal degradation by a pure culture of P. putida spi3 strain, in batch culture and using a synthe tic wastewater. A continuous ly stirred tank reactor fed with the synthetic wastewater was also operated and the bioreactor’s performance and robustness, when exposed to thiomersal shock loads, were evaluated. Finally, a bioreactor for the biological treatment of a real va ccine production effluent was set up and operated at different dilution rates. Thus it was possible to treat a real thiomersal-contaminated effluent, lowering the outlet mercury concentration to values below the European limit for mercury effluent discharges.

In Situ Near-Infrared (NIR) Versus High-Throughput Mid- Infrared (MIR) Spectroscopy to Monitor Biopharmaceutical Production

The development of biopharmaceutical manufacturing processes presents critical constraints, with the major constraint being that living cells synthesize these molecules, presenting inherent behavior variability due to their high sensitivity to small fluctuations in the cultivation environment. To speed up the development process and to control this critical manufacturing step, it is relevant to develop high-throughput and in situ monitoring techniques, respectively. Here, high-throughput mid-infrared (MIR) spectral analysis of dehydrated cell pellets and in situ near-infrared (NIR) spectral analysis of the whole culture broth were compared to monitor plasmid production in recombinant Escherichia coil cultures. Good partial least squares (PLS) regression models were built, either based on MIR or NIR spectral data, yielding high coefficients of determination (R-2) and low predictive errors (root mean square error, or RMSE) to estimate host cell growth, plasmid production, carbon source consumption (glucose and glycerol), and by-product acetate production and consumption. The predictive errors for biomass, plasmid, glucose, glycerol, and acetate based on MIR data were 0.7 g/L, 9 mg/L, 0.3 g/L, 0.4 g/L, and 0.4 g/L, respectively, whereas for NIR data the predictive errors obtained were 0.4 g/L, 8 mg/L, 0.3 g/L, 0.2 g/L, and 0.4 g/L, respectively. The models obtained are robust as they are valid for cultivations conducted with different media compositions and with different cultivation strategies (batch and fed-batch). Besides being conducted in situ with a sterilized fiber optic probe, NIR spectroscopy allows building PLS models for estimating plasmid, glucose, and acetate that are as accurate as those obtained from the high-throughput MIR setup, and better models for estimating biomass and glycerol, yielding a decrease in 57 and 50% of the RMSE, respectively, compared to the MIR setup. However, MIR spectroscopy could be a valid alternative in the case of optimization protocols, due to possible space constraints or high costs associated with the use of multi-fiber optic probes for multi-bioreactors. In this case, MIR could be conducted in a high-throughput manner, analyzing hundreds of culture samples in a rapid and automatic mode.

In situ NIR spectroscopy monitoring of plasmid production processes effect of producing strain, medium composition and the cultivation strategy

BACKGROUNDWhile the pharmaceutical industry keeps an eye on plasmid DNA production for new generation gene therapies, real-time monitoring techniques for plasmid bioproduction are as yet unavailable. This work shows the possibility of in situ monitoring of plasmid production in Escherichia coli cultures using a near infrared (NIR) fiber optic probe. RESULTSPartial least squares (PLS) regression models based on the NIR spectra were developed for predicting bioprocess critical variables such as the concentrations of biomass, plasmid, carbon sources (glucose and glycerol) and acetate. In order to achieve robust models able to predict the performance of plasmid production processes, independently of the composition of the cultivation medium, cultivation strategy (batch versus fed-batch) and E. coli strain used, three strategies were adopted, using: (i) E. coliDH5 cultures conducted under different media compositions and culture strategies (batch and fed-batch); (ii) engineered E. coli strains, MG1655endArecApgi and MG1655endArecA, grown on the same medium and culture strategy; (iii) diverse E. coli strains, over batch and fed-batch cultivations and using different media compositions. PLS models showed high accuracy for predicting all variables in the three groups of cultures. CONCLUSIONNIR spectroscopy combined with PLS modeling provides a fast, inexpensive and contamination-free technique to accurately monitoring plasmid bioprocesses in real time, independently of the medium composition, cultivation strategy and the E. coli strain used.

Enrichment of a PHA producing microbial community in a continuous bioreactor setup

Polyhydroxyalkanoates (PHAs) are biosynthetic polyesters, biodegradable and biocompatible making them of great interest for industrial purposes. The use of low value substrates with mixed microbial communities (MMC) is a strategy currently used to decrease the elevated PHA production costs. PHA production process requires an important step for selection and enrichment of PHA-storing microorganisms which is usually carried out in a Sequencing Batch Reactor (SBR). The aim of this study was to optimize the PHA accumulating culture selection stage using a 2-stage Continuous Stirrer Tank Reactor (CSTR) system. The system was composed by two separate feast and famine bioreactors operated continuously, mimicking the feast and famine phases in a SBR system. Acetate was used as carbon source and biomass seed was highly enriched in Plasticicumulans acidivorans obtained from activated sludge. The system was operated under two different sets of conditions (setup 1 and 2), maintaining a system total retention time of 12 hours and an OLR of 2.25 Cmmol/L.h-1. An average PHB-content of 3.3 % wt was obtained in setup 1 and 4.8% wt in setup 2. Several other experiments were performed in order to better understand the continuous system behaviour, using biomass from the continuous system. With the fed-batch experiment a maximum of 8.1% PHB was stored and the maximum substrate uptake and specific growth rates obtained in the growth experiment (1.15 Cmol Cmol-1.h-1 and 0.53 Cmol Cmol-1.h-1) were close to the ones from continuous system (1.12 Cmol Cmol-1.h-1 and 0.59 Cmol Cmol-1.h-1). The microbial community was characterized trough microscopic visualization, Denaturing Gradient Gel Electrophoresis (DGGE) analysis and Fluorescent in situ hybridization (FISH). The last studied performed mimicked the continuous system by building up a SBR system with all the same operational conditions while adding an extra acetate dosage during the 12 h cycle, simulating the substrate passing from the feast to the famine reactors under continuous operation. It was shown that possibly the continuous system was not able to efficiently select for PHB storing organisms under the operational conditions imposed, although the selected culture was capable of consuming the substrate and grow fast. This main conclusion might have resulted from two major factors affecting the system performance: the ammonium concentration in the Feast reactor and the amount of substrate leaching from the Feast to the Famine reactor.

Cell functional enviromics: applications to Pichia pastoris

The present PhD thesis develops the cell functional enviromics (CFE) method to investigate the relationship between environment and cellular physiology. CFE may be defined as the envirome-wide cellular function reconstruction through the collection and systems-level analysis of dynamic envirome data. Throughout the thesis, CFE is illustrated by two main applications to cultures of a constitutive P. pastoris X33 strain expressing a scFv antibody fragment. The first application addresses the challenge of culture media development. A dataset was built from 26 shake flask experiments, with variations in trace elements concentrations and basal medium dilution based on the standard BSM+PTM1. Protein yield showed high sensitivity to culture medium variations, while biomass was essentially determined by BSM dilution. High scFv yield was associated with high overall metabolic fluxes through central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy-generating pathways. CFE identified three cellular functions (growth, energy generation and by-product formation) that together described 98.8% of the variance in observed fluxes. Analyses of how medium factors relate to identified cellular functions showed iron and manganese at concentrations close to PTM1 inhibit overall metabolic activity. The second application addresses bioreactor operation. Pilot 50 L fed-batch cultivations, followed by 1H-NMR exometabolite profiling, allowed the acquisition of data for 21 environmental factors over time. CFE identified five major metabolic pathway groups that are frequently activated by the environment. The resulting functional enviromics map may serve as template for future optimization of media composition and feeding strategies for Pichia pastoris. The present PhD thesis is a step forward towards establishing the foundations of CFE that is still at its infancy. The methods developed herein are a contribution for changing the culture media and process development paradigm towards a holistic and systematic discipline in the future.