Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield
© Routledge et al; licensee BioMed Central Ltd. 2011
Received: 30 September 2010
Accepted: 22 March 2011
Published: 22 March 2011
Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.
Addition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of P. pastoris increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD595-1) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (k L a) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium.
We show that addition of a range of antifoaming agents to shake flask cultures of P. pastoris increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture.
The laboratory-scale production of recombinant proteins using P. pastoris requires that cells are cultured either in large shake flasks or in continuously stirred tank bioreactors. In these vessels, the formation of foam is an issue that requires intervention. This is in contrast to the situation in the small vessels typically used in the initial stages of protein production experiments where foaming is minimal .
Foaming can lead to reduced yields since bursting bubbles can damage proteins  and can also result in a loss of sterility if the foam escapes . In bioreactors, foaming can lead to over-pressure if a foam-out blocks an exit filter. To prevent the formation of foam, mechanical foam breakers, ultrasound or, most often, the addition of chemical antifoaming agents (or "antifoams")  are routinely employed.
There is a well-established literature on antifoams . One useful classification categorizes them as either hydrophobic solids dispersed in carrier oil, aqueous suspensions/emulsions, liquid single components or solids [4–6]. Several mechanisms of action for these agents have been suggested which include bridging-dewetting, spreading fluid entrainment and bridging-stretching . Many are commercially-available, with 19 being sold by Sigma-Aldrich alone. While little information is routinely given about their composition, their specific antifoam properties have been thoroughly investigated. These include their effects on foam height with time, their influence on the volumetric oxygen mass transfer coefficient (kLa) of the system, their gas hold-up characteristics and their globule size and distribution in relation to their action upon foams [3, 5, 7–11]. Such studies have been performed in various growth media in both the absence and presence of cultures of prokaryotic and eukaryotic microbes.
In contrast, literature on the biological effects of antifoams on recombinant protein yields from microbial cell factories is more limited. Additional file 1: Table S1 shows an analysis of representative examples of this body of work including previous studies on four bacterial hosts and one yeast species. In some cases, the additives tested are not antifoams sensu stricto. It is also noteworthy that the yeast, Schizosaccharomyces pombe, is not widely used in biotechnology applications and that there have been no prior studies on the biological effects of antifoam addition to recombinant P. pastoris cultures. A recent review stated that in the last 15 years, 80% of all recombinant genes reported in the literature were expressed in either Escherichia coli or P. pastoris. In this study, we therefore examined five antifoams that are widely used in controlling the foaming of recombinant P. pastoris cultures [13–16] in order to analyze effects over and above that of their de-foaming action. We looked at polypropylene glycol (PPG) P2000 that is analogous to previously-examined liquid single components of the PPG-type  as well as examples from other categories such as Antifoam A and Antifoam C, which are silicone polymers, SB2121, which is a polyalkylene glycol, and J673A, which is an alkoxylated fatty acid ester on a vegetable base and has not previously been documented in this context: for all antifoams examined, this was the first report of their effect on the yield of recombinant GFP secreted from shake-flask cultures of P. pastoris.
We wanted to establish whether antifoams affect recombinant protein yield in P. pastoris X33 cultures, and if so to investigate the underlying mechanisms. To examine this we chose an experimental system, under the control of the methanol-inducible AOX1 promoter, comprising GFP secreted from 20 mL cultures in shake flasks in the presence of five different antifoams at a range of concentrations from 0-1% v/v. These concentrations are higher than the 0.1% routinely used for de-foaming purposes. The total amount of GFP in these 20 mL cultures (the total yield) was measured by fluorimetry 48 h post-induction.
Antifoam addition affects total GFP yield in shake flasks
The effects of antifoam addition are due to changes in culture density for P2000 and SB2121
To account for any changes in the growth characteristics of the cells on addition of the antifoams, we normalized the total yield to the optical density of the cultures to obtain the specific yield (μg OD595-1). OD595 was demonstrated to be a reliable measure of cell density in these experiments by comparing the number of cells at a given OD595 in the absence and presence of a range of concentrations of the different antifoams used in our study: there was no statistically significant difference in cell number between cells harvested at a given OD595 in the absence or presence of all antifoam concentrations tested. Typical values were 4.8 × 107 cells/mL at an OD595 of 20.5 in the absence and presence of 0.5% SB2121.
For Antifoam A, Antifoam C and J673A, the specific yield data were similar in trend to the total yield data (Figure 1A-C): addition of these antifoams in the range 0.6-1% v/v caused a significant increase in specific yield compared to the control cultures with no antifoam. For cultures containing P2000 or SB2121, however, there was no statistically significant difference in the specific yield at each antifoam concentration compared with the control except for 1% SB2121 where P < 0.05 (Figure 1D-E). This suggested that the enhancements in total yield due to P2000 or SB2121 addition might be attributable to changed growth characteristics of the cells. The specific growth rates (μ) for cultures containing either 1% P2000 or 1% SB2121 were 0.17 h-1 and 0.18 h-1 respectively compared with 0.17 h-1 for the control samples (0% antifoam) indicating that the growth characteristics during the log phase were not affected by the presence of the antifoams. However, we noted an increase in OD595 (at both 24 and 48 h) with increasing antifoam concentration for both antifoams (Figure 1D-E; 48 h data), which was less pronounced for Antifoam A, Antifoam C and J673A (Figure 1A-C). We concluded, therefore, that there was more than one mechanism of antifoam action: one due to changed culture density (P2000, SB2121) and a second due to increased cellular production levels of recombinant GFP (Antifoam A, Antifoam C, J673A).
Antifoam addition does not affect cell viability
The foam destruction capacity of an antifoam is related to its ability to improve GFP yield
Improved yields cannot be explained by antifoam-induced changes in GFP fluorescence
In order to determine whether any of the antifoams affected the fluorescence of GFP, 1% antifoam was incubated in BMMY for 48 h to mimic the experimental set-up. This was then spiked with a similar concentration of recombinant GFP standard to that obtained in Figure 1. There was no significant difference between the fluorescence of GFP in the presence and absence of any of the antifoams suggesting that they did not influence the sample readings. The fluorescence values of the antifoams themselves were also measured at 1% and found to be minimal, similar to the buffer control readings.
The k L a characteristics of antifoam-containing cultures are not correlated with improvements in GFP yield
DO in shake flasks is not affected by the presence of antifoams P2000 and SB2121
Addition of Antifoam A, Antifoam C or J637A affects the total yield of GFP secreted into the medium
Antifoams have previously been suggested to alter the growth of cells and influence protein yield in bioprocesses [2, 11], but their addition to P. pastoris cultures has never been examined systematically. In this study, addition of the five antifoams tested increased the total yield of secreted recombinant GFP produced by 20 mL P. pastoris cultures. Generally, the total GFP yield secreted into the culture medium was increased when antifoam was added at concentrations of at least 0.4% v/v compared with the 0% antifoam control. Addition of 1% J673A to a 20 mL culture yielded 394 μg GFP compared with 246 μg GFP when there was no antifoam present. This is notable as J673A is approved for industrial use.
Antifoams can be split into two categories of fast and slow antifoams, depending on their mechanism of foam destruction. Slow antifoams are often oils which destroy foam over a longer period of time. Fast antifoams, as examined in this study, are generally mixed agents which enter the foam film and destroy it by a bridging-stretching mechanism . It has also been observed that the most effective agents at destroying foam are those with the most efficient oil film spreading characteristics . The least effective de-foaming agents in this study were Antifoams A and C, which are 30% aqueous emulsions of Antifoam A concentrate . Their reduced de-foaming capability was accompanied by the weakest ability to increase the yield of protein. In contrast, J673A addition produced one of the best results, almost doubling the yield. This antifoam is an alkoxylated fatty acid ester on a vegetable oil base and it is known that vegetable oils can alter the structure of foams by increasing bubble size and reducing the stability of the foam . While vegetable oils may be metabolized as a carbon source , which might explain why J673A addition enhanced the yield of GFP, our data show that J673A did not influence the growth of the cells (Figure 1C), but rather enhanced the amount secreted into the medium (Figure 6). J673A was additionally found to be the most effective de-foamer of the panel of five antifoams that we assayed (Figure 3).
Antifoams are also known to affect the k L a of a system, which can be influenced by several factors such as medium viscosity, the presence of organisms and their by-products. These variables affect both k L (ms-1) and a (specific surface area m-1) [24, 25]. For example, antifoam addition is known to have an effect  by enhancing bubble coalescence and increasing bubble size which leads to a reduction in the specific surface area thereby lowering k L a[3, 8, 24, 25, 27]. However, it has also been previously observed [25, 28] that at higher concentrations of antifoam the k L a rises possibly due to the detrimental effects of bubble coalescence. Consequently the reduced specific surface area (a) reaches a limit and bubbles coalesce suppressing surface motility and decreasing surface tension. This then leads to decreasing bubble size and k L a rises again. Additionally it is possible that antifoams accumulate oxygen from rising bubbles, as they have good oxygen solubility, and then release it to the aqueous phase. Bubbles bursting at the surface disperse small drops of antifoam causing more oxygen to be released [25, 28]. In the case of oils which have a greater oxygen solubility than water, oil droplets may increase oxygen permeability in the water boundary layer of the gaseous dispersion . Yagi and colleagues suggested that surfactants can cause rippling or eddying which influences the k L a. They also found that k L was not greatly affected by antifoams, but that their main effect was on a. Koide subsequently suggested that the ability of antifoams to reduce k L is less for bubble swarms than for a single bubble .
We found that in shake flasks, the k L a was higher at mid-range values and decreased with increasing concentration, but that there was no statistically significant correlation between increased k L a and total yield. It is therefore possible that the k L a is already sufficient for the cells to grow and produce protein and is not a limiting factor, or that a combination of factors is responsible for the increases in total yield that we observed. This is supported by the DO shake flask data which suggest there is no difference between the DO in flasks without antifoam and those with either P2000 or SB2121 added. Combining flow cytometry and fluorimetry data showed that the antifoams can influence the amount of GFP retained inside the cell as well as the amount secreted into the medium. Antifoam A, Antifoam C and J673A enhanced the GFP secreted compared to 0% antifoam suggesting that the increase in total yield observed could be due to this secretion effect. This is consistent with an earlier study which suggested that antifoams can affect cell permeability in yeast by perturbing sterol biosynthesis which then alters the permeability of the membrane . This is currently under investigation.
We show that when Antifoam A, Antifoam C, J673A, P2000 or SB2121 are added at concentrations higher than those routinely used for de-foaming purposes, they all increase the total yield of shake-flask cultures. Two effects are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture. Addition of commonly-used antifoaming agents to shake flask cultures of yeast is therefore an effective way to increase the total yield of the recombinant protein being produced; any necessary changes to downstream steps such as protein purification are therefore likely to be worthwhile. Furthermore, this study provides insight into the manner in which antifoams interact with microbial cell factories: any data contributing to a fuller understanding of the specific effects of an antifoam on the growth and yield characteristics of such cultures, in addition to its primary action as a de-foamer, will be essential in bioprocess optimisation. These findings should provide an impetus to increase productivity in shake flask cultures of P. pastoris.
Yeast strains and culturing conditions
Pichia pastoris strain X33 transformed with pPICZαA-GFPuv (designated X33GFPuv)  was used in all experimental procedures. Cells were cultured in shake flasks in medium buffered to pH 6.0 with 1 M potassium phosphate buffer and at 30 °C and 220 rpm. BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 4 × 10-5% biotin, 1% glycerol ) was used for the initial biomass accumulation stage before transferring to the induction medium, BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% YNB, 4 × 10-5% biotin, 0.5% methanol ), to induce production of GFP.
Shake flask cultures
Cells were cultured in 50 mL BMGY in 250 mL baffled shake flasks to accumulate biomass. 20 mL BMMY was then inoculated to a final OD595 of 1.0 and transferred to a 100 mL non-baffled shake flasks for antifoam evaluations. Each evaluation was done in triplicate, with each flask containing the desired concentration of antifoam (0%, 0.2%, 0.4%, 0.6%, 0.8% or 1.0% (v/v)) with incubation at 30 °C and 220 rpm. After 24 h, 100% sterile methanol was added to 1% v/v to maintain production of GFP . All optical density measurements were blanked against the relevant antifoam-containing medium. Since the antifoams themselves might influence OD595, we analyzed the relationship between OD595 readings in the absence and presence of a range of concentrations of different antifoams. In all cases the pair wise relationship was linear (R2 was 0.91-0.99). We further verified that OD595 was a reliable measure of cell density by comparing the number of cells at a given OD595 in the absence and presence of a range of concentrations of different antifoams. There was no statistically significant difference in cell number between cells harvested at a given OD595 in the absence or presence of any of these antifoam concentrations, suggesting that OD595 is indeed a robust measurement of cell density.
The antifoams tested in this study were Schill and Schelinger's Struktol SB2121 (a polyalkylene glycol), Schill and Schelinger's Struktol J673A (an alkoxylated fatty acid ester on a vegetable base), Fluka P2000 (a polypropylene glycol), Sigma Antifoam A (a 30% emulsion of silicone polymer) and Sigma Antifoam C (a 30% emulsion of silicone polymer). All antifoams were autoclaved prior to use and each shake flask experiment was performed in triplicate, with the undiluted antifoam being added directly to the medium.
Culture supernatants (100 μL) were assayed at 24 h and 48 h post-induction for GFP fluorescence using a Spectramax Gemini XS plate reader with an excitation wavelength (λexe) of 397 nm, and emission wavelength (λem) of 506 nm. Triplicate determinations were performed for each independent sample. All samples and blanks were buffered to pH >7.0 using 50 μL 1 M potassium phosphate pH 7.5. Data were collected at 25 °C. To determine the concentration of GFP in each of the samples, a recombinant GFP standard (Vector Laboratories Ltd) was used to construct a standard curve relating RFU to protein concentration, as previously described . All data were analyzed using a one-way ANOVA to test for a significant difference between any of the means. In all cases P < 0.001 indicating a high degree of significance. A Dunnett's multiple comparison test was then performed to compare each treatment mean (addition of various antifoam concentrations) and the control mean (0% antifoam).
Total protein analysis
The total protein content of culture supernatants (2 μl) at 48 h post-induction was analyzed by bicinchoninic acid (BCA) assay. Cultures were examined in the presence of representative concentrations of 0%, 0.5% and 1% antifoam. 4.9 mL of proprietary BCA solution (B9643, Sigma) was mixed with 100 μL 4% mM copper (II) sulfate solution (C2284, Sigma). 200 μL of this solution was used to assay each independent supernatant sample in duplicate using a plate reader (BioTek Instruments) at 570 nm. To determine the concentration of protein in the samples, a bovine serum albumin standard (Sigma) was used to plot a standard curve. The data were analyzed using a one-way ANOVA (P < 0.0001) and a Dunnett's multiple comparison test.
Bartsch antifoam test
Bartsch tests were conducted following a protocol adapted from that outlined by Denkov and colleagues . A 500 mL graduated glass cylinder was filled with 166 mL BMMY medium and in all cases except for the control, antifoam was added to 0.01% v/v. The cylinder was sealed with parafilm and shaken ten times at ambient temperature. The height of the foam was recorded using the graduations on the cylinder every 30 s for 15 min. Determinations were performed in quintuplet for each antifoam. The activity of a given antifoam was reported as a volume , obtained by subtracting the volume of medium from the total volume (foam plus medium) in the cylinder.
k L a determination
Dissolved oxygen measurements
Dissolved oxygen was measured in 125 mL non-baffled shake flasks with DO fluorescent sensors (PreSens) attached to the underside of each flask. The flasks were placed on a shake flask reader which excites the dyes in the sensors and allows the DO data to be logged over 48 h with SFR software (Presens).
Shake flask cultures of P. pastoris, as described above, were used to generate samples for flow cytometry analysis. The antifoams used were Antifoam A at 0.6%, Antifoam C at 0.6%, J673A at 0.8%, P2000 at 0.6% and SB2121 at 0.6% (v/v). Triplicate flasks were used for each antifoam. 48 h samples were diluted 1:1000 in phosphate buffered saline to a final concentration of 106-107 cells mL-1, as determined using a haemocytometer. Fluorescent measurements were made using a Beckman Coulter (High Wycombe, UK) flow cytometer with λexe = 488 nm from an argon-ion laser at 15 mW. Diluted samples were additionally stained with 10 μL propidium iodide (PI; 1 mgmL-1 in water). All solutions were passed through a 0.2 μm filter, immediately prior to use, to remove particulate contamination. The optical filters were set up so that PI fluorescence was measured at 630 nm and GFP fluorescence was measured at 525 nm. The data were analyzed using a one-way ANOVA (P < 0.0001) and a Dunnett's multiple comparison test.
This work was supported by an EPSRC CASE award with Applikon Biotechnology Ltd supporting SJR. We thank the Aston Research Centre for Healthy Ageing (ARCHA) for support and Dr William Holmes (MedImmune), Dr Andrew Devitt (Aston University) and Dr Sharon Brownlow (Applikon Biotechnology) for helpful comments and suggestions.
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