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Microaerophilic environments improve the productivity of medium chain length polyhydroxyalkanoate biosynthesis from fatty acids in Pseudomonas putida LS46
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Microaerophilic environments improve the productivity of medium chain length polyhydroxyalkanoate biosynthesis from fatty acids in Pseudomonas putida LS46 ⁎
Warren Blunta, , Christopher Dartiailha, Richard Sparlingb, Daniel Gapesc, David B. Levina, Nazim Ciceka a b c
Department of Biosystems Engineering, University of Manitoba, Winnipeg, Manitoba, R3T 5V6, Canada Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada Scion Research, Te Papa Tipu Innovation Park, 49 Sala Street, Private Bag 3020, Rotorua, 3046, New Zealand
A R T I C L E I N F O
A B S T R A C T
Keywords: Polyhydroxyalkanoates Pseudomonas putida Dissolved oxygen Bioreactor Biopolymer Microaerophilic
Medium chain length polyhydroxyalkanoate (mcl-PHA, a commercially desirable biopolymer) synthesis from fatty acids is an oxidative process. Growth and mcl-PHA synthesis were studied in Pseudomonas putida LS46 when subjected to a spectrum of oxygen transfer rates, which were intended to simulate dissolved oxygen (DO) gradients likely to be encountered during process scale-up. It was found that maintaining DO at 1–5% (of air saturation at 30 °C) initiated significant mcl-PHA synthesis and concurrent growth at < 0.5 μmax was also observed. Further reductions in DO resulted in nearly negligible growth, but improved biopolymer synthesis. When the volumetric oxygen mass transfer coefficient (kLa) was reduced to 38 h−1, DO was below detectable limits and both the biopolymer content (57.3 ± 3.8% CDM) and yield (0.62 ± 0.06 g mcl-PHA g octanoic acid−1) reached the maximum observed values. Under these conditions, the maximum rate of polymer synthesis was also observed (227.9 ± 9.8 mg PHA g residual cell mass−1 h−1), and this value was higher than that which could be achieved using nitrogen limitation. Further reductions in oxygen transfer rate did not improve productivity, and PHA synthesis completely ceased when aeration was replaced with N2 gassing. These results suggest that manipulation of the bioreactor oxygen transfer rate during the PHA accumulation phase could be a strategy used to improve overall productivity during a fed batch or continuous feed process.
1. Introduction Medium chain length polyhydroxyalkanoates (mcl-PHAs) are C6-C14 polyesters synthesized by bacteria and have potential value for diverse set of applications [1,2]. Currently production costs are too high for economically viable, commercial scale mcl-PHA production [3]. Development and subsequent scale up of production strategies targeting high overall productivity (a function of reaction rates, yield, and product titer) can help reduce costs and secure the future of mcl-PHA as a commercially valuable biopolymer [4,5]. To synthesize a significant amount of mcl-PHA, most bacteria require nutrient imbalance and to achieve this, medium components like carbon, nitrogen, or phosphate are added in growth-limiting quantities [6]. During mcl-PHA production and many strictly aerobic bioprocesses, gas-to-liquid mass transfer of oxygen is often rate limiting and has a significant impact on the overall productivity [7–9]. Several
studies have supplemented the aeration medium with pure oxygen to increase the driving force for oxygen mass transfer [7,10–12]. Operating bioreactors at elevated pressure has also been used to increase the driving force, although increased pressure has also been shown to have a negative impact on the oxygen mass transfer coefficient, kLa [13,14]. Both strategies are aimed at prolonging the growth phase before oxygen mass transfer becomes rate limiting to achieve higher cell densities and increased volumetric productivity. However, the effect that low dissolved oxygen (DO) environments, likely to be encountered in largerscale and high cell density cultivations, have on mcl-PHA synthesis has not been considered in detail. Low DO conditions have been reported to increase the intracellular scl-PHA content in several microorganisms grown on a variety of substrates [15–17]. Using mixed microbial cultures, Pratt et al. observed increased yield during the early stages of growth in low DO environments, but the rate of accumulation was lower than in high DO environments [18]. Some studies have also
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Corresponding author at: E2-376 Engineering and Information Technology Complex (EITC), 75A Chancellor’s Circle, University of Manitoba, Winnipeg, MB R3T 5V6, Canada. E-mail addresses: [email protected], [email protected] (W. Blunt), [email protected] (C. Dartiailh), [email protected] (R. Sparling), [email protected] (D. Gapes), [email protected] (D.B. Levin), [email protected] (N. Cicek). http://dx.doi.org/10.1016/j.procbio.2017.04.028 Received 20 September 2016; Received in revised form 5 January 2017; Accepted 9 April 2017 1359-5113/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Blunt, W., Process Biochemistry (2017), http://dx.doi.org/10.1016/j.procbio.2017.04.028
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reported changes in the monomeric composition of both scl-and mclPHA when the culture was subject to low DO environments [19,20]. Given that the cellular redox state has been linked to mcl-PHA synthesis [21], oxygen availability and the redox state of the substrate could play a role in mcl-PHA accumulation. Structurally related substrates (alkanes, alkanoic acids) are highly reduced and are preferred for mcl-PHA synthesis due to the high yield [7,10,12]. Production of mcl-PHA from such substrates requires intermediates of the βoxidation cycle [22]. This is an oxidative process (in contrast to synthesis of PHB from glucose) in which FAD and/or NAD+ are used as electron acceptors, and these must be regenerated by electron transport to O2. Therefore, mcl-PHA synthesis from related (highly reduced) substrates under oxygen-limited conditions would be a phenomenon of interest. Little information is available regarding how mcl-PHA production (from fatty acids or other substrates) responds to low DO environments, particularly compared to short chain length (scl) PHA production. Several studies have reported improved scl-PHA polymer production from carbohydrate substrates in low DO environments [15–17]. In this work, we have used a scaled-down approach to assess mclPHA production rates in low DO environments using Pseudomonas putida LS46 grown on octanoic acid in bench scale bioreactors operated in batch mode. The objective of this study was to understand how oxygen mass transfer limitations affect growth and mcl-PHA biosynthesis from fatty acids.
Table 1 Summary of various conditions tested, with DO expressed as a percent of air saturation (AS), and as concentration (mg L−1 and μM) for comparability. Reactor operating parameters required to maintain these conditions, including airflow rate, agitation rates, and resulting volumetric oxygen mass transfer coefficient, are also shown. A minimum of two biological replicates was performed for each condition. Dissolved oxygen content
Vol. O2 Mass Transfer Coefficient, kLa
Air Flow Rate
Mixing Cascade
% AS (30 °C)
mg L−1 O2
μM O2
hour−1
LPM
rpm
0 0 1 5 10 40
0.02a 0.04a 0.07b 0.34b 0.68b 2.71b
0.53 1.28 2.12 10.58 21.16 84.65
38 78 78 78 78 78
3 6 6 6 6 6
250 250 250–410 250–620 250–740 250–900
< < < <
kLa kLa kLa kLa
< < < <
145 310 540c 1016c
a
Reported results were measured with a MIMS. Results measured with electrochemical DO probe. kLa values > 310 h−1 could not be reliably measured due to insufficient response time of probe. Values > 310 h−1 were estimated by extrapolating the trend from the range that could be reliably measured with the probe. b c
flow rate was held constant in all experiments at either three liters per minute (LPM), equal to one volume air per liquid volume per minute (VVM), or six LPM (2 VVM). A mixing cascade (250–900 rpm) was used to maintain DO at levels above detectable limits (non-zero readings with the DO electrode). Since some of the conditions tested were below the threshold of detection of the DO electrode, these were characterized by the oxygen mass transfer coefficient (kLa), which was measured via the dynamic out-gassing method. It was assumed that the probe response could be modeled as first order [25]. Due to insufficient response time of the DO electrode, kLa could not be reliably measured for kLa > 310 h−1, which corresponded to mixing rates of approximately 600 rpm. An HPR-40 dissolved species membrane-inlet mass spectrometer (MIMS) equipped with a HAL 201 RC quadrupole mass analyzer and Faraday Cup detector (Hiden Analytical, Warrington, United Kingdom) was also used to estimate DO concentrations for conditions below the detection threshold of the DO electrode. A summary of the oxygen transfer conditions tested is presented in Table 1. It was assumed that the oxygen solubility in the culture medium at 30 °C was 6.77 mg L−1, which was measured previously in a minimal medium [26].
2. Materials and methods 2.1. Microorganism, medium, and substrates The production strain used in this study was Pseudomonas putida LS46 [23]. Glycerol stock cultures were stored at −80 °C, and were periodically revived and maintained on agar plates stored at 4 °C. The medium used was Ramsay’s medium [24], with 20 mM octanoic acid added as a carbon source. Baffled flasks (500 mL flask with 100 mL working volume) were used for the reactor inoculum and were incubated at 30 °C and 150 rpm shaking. The initial pH of the medium in flask cultures was adjusted to 7.0 with the addition of NaOH. The medium for bioreactor experiments was slightly modified by increasing the concentration of (NH4)2SO4 to 11.35 mM (1.5 g L−1) to ensure excess ammonium throughout each trial. 2.2. Reactor setup and preparation All reactor experiments were conducted in a 7 L glass, round-bottom reactor with a 3 L working volume. The reactor was equipped with a Rushton impeller, three baffles, a polarographic DO electrode, and a pH electrode. All reactor equipment was purchased from Applikon Biotechnology (Foster City, CA). The oxygen transfer capability of the reactor was improved through the addition of two Pyrex microbubblers, which were connected to the existing sparging tube with Masterflex LS25 tubing (4.8 mm inner diameter), and located underneath the impeller. The medium, substrate, and 0.2 mL of a silicone antifoaming agent (obtained from Husky Energy in Minnedosa, MB) were added to the reactor prior to autoclaving at 121 °C for 40 min. After cooling, the reactor was stabilized with air overnight to allow polarization of the DO electrode, and maintained at 30 °C. Prior to addition of the inoculum, a one-point calibration of the DO electrode was performed at operating air flow rate, and this condition was assigned a value of 100% of air saturation (AS). The pH was adjusted to a set point of 6.5 via addition of 1 M HCl and 1 M NaOH through automated peristaltic pumps.
2.4. Reactor monitoring Experiments were initiated with the addition of 1% (vol−1) inoculum. At this time the medium was saturated with air, (DO = 100%) and the DO was allowed to fall until the indicated set point was reached. A 40 mL sample of culture was taken in 1–2 h intervals from the reactor for analysis of biomass, PHA, residual octanoic acid, and residual free ammonia. These procedures are described below.
2.5. Measurement techniques 2.5.1. Total biomass and residual (PHA-free) biomass Each sample from the reactor was transferred into pre-weighed 50 mL centrifuge tubes, and the total biomass was immediately separated from the supernatant by centrifugation (4000g for 20 min at 4 °C). The supernatant was stored at −20 °C. The pellet was then oven dried at 60 °C until no further loss in mass was detected (24 h), and then total biomass was determined gravimetrically. Residual (PHAfree) biomass was estimated as the difference between total biomass and the mass of detected PHA monomers.
2.3. Oxygen transfer conditions Air was used for sparging the culture in all studies. The volumetric 2
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cells had grown to an average cell density of 0.7 g L−1 CDM. This portion of the growth curve (after 6 h) was used to calculate the specific growth rates. Fig. 2 shows the change in specific growth rate at different DO conditions. Due to the lack of sensitivity of the bioreactor probe in periods when the DO was not controlled (constant mixing at 250 rpm and constant aeration at either 3 LPM or 6 LPM), the MIMS was used to confirm the DO concentration in these two conditions (Table 1). The relationship between growth rate and DO concentration followed Monod kinetics (R2 = 0.99) and the fitted parameters indicated μmax = 0.57 h−1 and the oxygen half saturation constant (Ks) to be 0.19 mg L−1 (2.7% AS at 30 °C). This Ks value is relatively low compared to the values reported for other Pseudomonas species and strains and indicates a high affinity for oxygen [30,31]. A possible explanation for this may be the presence of two putative subunits for a cytochrome bd complex in the genome annotation for the respiratory chains in P. putida LS46 (www.img.jgi.doe.gov). Cytochrome bd is a terminal oxidoreductase with a very high affinity for oxygen (but lower ATP yield) and is expressed in several microorganisms in stressful environments, including microaerophilic conditions [32,33]. Tseng et al. [34] have shown that cytochrome bd expression increases dramatically when DO falls below 10% AS, and reached a maximum at 0% AS in E. coli cultures. Based on the relatively steep reduction in growth rate at DO < 10% AS in P. putida LS46, it may be possible that this also occurs in LS46. Proteomic analysis would be necessary to confirm this. Overall, the data indicates that P. putida LS46 is capable of not only survival, but also growth in low DO environments.
2.5.2. mcl-PHA and fatty acid analysis Preparation and analysis of the dried biomass samples for cellular mcl-PHA content followed the acid-catalyzed methanolysis procedure previously described [3]. The same procedure was followed for analysis of octanoic acid in the culture medium, except that 1 mL of thawed culture supernatant (allowed to come to room temperature and vortexed thoroughly) was added to each reaction vial. Determination of mcl-PHA content of the cells and octanoic acid was accomplished using an Agilent 7890A gas chromatograph equipped with a splitsplitless inlet (operated in split mode, split ratio 10:1), a DB23 capillary column (Agilent, 30 m × 250 μm × 0.25 μm), and a flame ionization detector. Method operating parameters and peak quantification are described elsewhere [3]. 2.5.3. Residual free ammonium Residual ammonium in the culture medium was determined via the Lachat QuikChem® Method 10–107-06-1-J. Briefly, this method uses a spectrophotometer coupled to a flow injection system (Lachat Instruments, CO) that measures the blue color change resulting from the reaction of alkaline phenol and sodium hypochlorite with ammonia to form indophenol blue, which is measured at 630 nm. 2.6. Productivity and yield calculations Volumetric productivity of a batch was calculated as the maximum value of PHA titer divided by the cultivation time at which it was observed. Specific productivity was calculated as the increase in PHA concentration in a given time divided by the mean residual biomass measured during that time. Yield was calculated as the slope of a regression of PHA titer (g L−1) and substrate uptake (g L−1). The maximum theoretical yield of mcl-PHA from octanoic acid was calculated as 0.98 g g−1 following the approach used by Weusthuis et al. with the assumption that the polymer synthesized from octanoic acid can be treated as a homopolymer of C8 monomers [27].
3.2. PHA accumulation The maximum cellular PHA content data (Fig. 3) shows that little PHA was detected for conditions in which DO was maintained at 10% AS or higher. A significant increase in cellular PHA content (up to 41.4% of cell dry mass, CDM) was consistentlyevident when DO was maintained at l% AS or lower. At the lowest OTR tested (kLa = 38 h−1), the final cellular mcl-PHA content was 57.3% CDM. The final cellular PHA content was somewhat variable (23 to 38% CDM) when the DO was maintained at 5% AS. This variability may suggest this condition could be close to a switching point from growth to PHA synthesis in response to stress from low DO. Due to fluctuations in the PIDcontrolled DO measurement, further resolution was not possible. It appears that little PHA is accumulated when DO > 5% AS and whilst at DO ≤ 5% AS, cells begin to accumulate a significant amount of PHA. On the basis of final PHA content these results indicate that growth in shaker flasks, with the same C/N ratio and low working volumes, is oxygen limited. The relationship between oxygen limitation and PHA accumulation was first established as a way for disposal of excess reducing power during synthesis of PHB from glucose in Azotobacter vinelandii [17]. Since there is no reductive step during synthesis of mcl-PHA from βoxidation intermediates, this cannot explain observation of mcl-PHA synthesis from fatty acids in response to low DO. High NAD(P)H/NAD (P)+ ratios have been linked to mcl-PHA synthesis from fatty acids [21,35], as well as non-related substrates [36]. This redox imbalance would likely occur at low DO and cause inhibition of key enzymes in the TCA cycle [37]. If PHA is produced from intermediates of the βoxidation cycle, conversion of either the enoyl-CoA monomers (via an R-specific enoyl-CoA hydratase, PhaJ) or (S)-3-hydroxyacyl-CoA monomers (via epimerase enzyme) to (R)-3-hydroxyacyl-CoA monomers requires only one oxidative step. In this step, the acyl-CoA dehydrogenase enzyme oxidizes acyl-CoA monomers to enoyl-CoA monomers using FAD as the electron acceptor [1,38–40]. Storage of these monomers as PHA (primarily C8 and C10 in P. putida) would generate fewer reduced electron carriers compared to complete oxidation. Completion of the β-oxidation cycle requires oxidation of 3-hydroxya-
2.7. Carbon dioxide measurement and carbon balancing Carbon dioxide was measured in the reactor headspace at a mass-tocharge ratio of 44 using the MIMS. Further detail on MIMS calibration and quantification of CO2 is given elsewhere [28]. A carbon balance at any time during the fermentation could be performed as shown in Eq. (1).
[6 × C6 + 8 × C8 + 10 × C10] + 4 × (moles residual biomass ) t
+ C Balance =
∫0
[CO2] dt 8 × (moles octanoic acid consumed ) (1)
Where C6, C8, C10 represent moles of the corresponding monomer subunits produced (determined by GC). It was assumed that elemental composition of residual biomass could be approximated by the formula C4H7O2N and as having a molecular mass of 101 g mol−1 [29]. For experiments in which CO2 measurements were not available, it was assumed that the left hand side of this equation was equal to 1, and the equation was rearranged to back-calculate CO2 production. 3. Results and discussion 3.1. Growth rate Fig. 1 shows sample growth curves for an oxygen-excess condition (Fig. 1A) as well as an oxygen-limited condition (Fig. 1B). Specific growth rates were assessed as the increase in residual (PHA-free) biomass once the target DO content was reached. The target DO was typically reached about 6 h after inoculation in each trial, when the 3
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Fig. 1. A) Growth curve for A) condition in which DO was maintained at 40% and B) condition in which DO was below detectable limits with the bioreactor probe (kLa = 38 h−1). Data includes total biomass (○), residual biomass (▯), PHA concentration (☐), and cellular PHA content (♦). The DO content over time is represented by the continuous line. Error bars indicate standard deviations between biological replicates.
cyl-CoA to 3-ketoacyl-CoA, which uses NAD+ as the electron acceptor. Furthermore, subsequent oxidation of acetyl-CoA (produced from each completion of the β-oxidation cycle) via the TCA cycle generates 3 NADH and 1 FADH2 [1,41]. Therefore, due to redox imbalance at low DO the cell may not be able to further metabolize the available acylCoA, instead leaving it accessible to the monomer offering enzymes for PHA synthesis. Thus, mcl-PHA synthesis from fatty acids when oxygen is limited cannot be a ‘sink’ for electrons that has been previously described in PHB synthesis [17], but may be a mechanism for storing electrons as PHA to maintain a balanced redox state. A similar assertion was also made for degradation of PHB to supply reducing power required to elicit an anti-oxidant stress response in P. putida 14-3 [42].
This is not the case for synthesis of scl-PHA or mcl-PHA from carbohydrate substrates, since both pathways include reductive steps. Although improved scl-PHA production from carbohydrate substrates in low DO environments has been reported [15–17], we did not observe any improvement in mcl-PHA production from P. putida LS46 cultured on crude glycerol using sequentially lower surface-area-to volume ratios in baffled shaker flasks (data not shown). Further investigation is required, but this result is in agreement with other studies of mclPHA synthesis from non-related substrates via de novo fatty acid synthesis [43–45].
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Fig. 2. Specific growth rate (μ) at indicated oxygen transfer rate. Values measured after 6 h of cultivation, or when DO had reached its intended steady state value. Error bars indicate the standard deviations between a minimum of two biological replicates. Since kLa measurements more adequately describe conditions below the detection limit of the bioreactor probe and DO measurements more adequately describe high-intensity mixing conditions where kLa may not be reliable due to the response time of the probe (kLa > 310 hour−1), the x-axis is labelled with both parameters.
3.3. Monomer composition of polymer
Table 2 Summary of observed monomer composition among the conditions tested. The monomer composition was measured as the mean of all samples taken after the DO had reached its target concentration (generally after 6 h cultivation). A minimum of two biological replicates was performed for each condition.
The monomer compositions of the polymer synthesized under the conditions tested are shown in Table 2. For conditions in which PHA was accumulated the composition was not significantly different in any case. For these cases, the mean monomer composition was 5.9 ± 0.9% C6, 92.3 ± 1.1% C8, and 1.9 ± 0.6% C10. However, after the exogenous carbon source was depleted, the C8 monomers appeared to be consumed first, causing an enrichment of C6 and C10 monomers and significantly different monomer distributions. This suggests that prolonged carbon limitation may cause inconsistent product quality, and the timing of harvest might be critical. Dissolved oxygen has been previously observed to play a role in altering the monomer composition in both scl-PHA and mcl-PHA production. Lefebvre et al. report that low DO (1–4% AS) caused more than a two-fold increase in yield of poly-(3-hydroxyvalerate) from propionate during production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose and propionate using C. necator as compared to conditions when the DO was maintained at 50–70% AS [20]. Fernández et al. report that during mcl-PHA production from crude oleic acid using P. aeruginosa, lowering the OTR caused significant increase of C8, C12:1 and the appearance of C14:2 monomers, while C10, C12, and C14:1 all decreased [19]. The major distinction between these two previous
Dissolved oxygen content
kLa
Monomer Composition (mol%)
% AS (30 °C)
hour−1
C6
0 (3 LPM air) 0 (6 LPM air) 1 5 10 40
38 78 78 78 78 78
6.0 6.0 5.6 4.8 6.1 6.3
< < < <
kLa kLa kLa kLa
< < < <
145 310 540 1016
C8 ± ± ± ± ± ±
1.1 0.4 1.2 0.4 1.2 0.3
92.5 91.9 92.4 93.2 92.3 91.6
C10 ± ± ± ± ± ±
0.8 0.5 2.1 0.1 0.8 0.4
1.4 2.0 1.6 2.0 1.6 2.8
± ± ± ± ± ±
0.4 0.6 0.3 0.3 0.4 0.9
studies and this work is that we observed lower OTRs to significantly improve biopolymer content and productivity. Different substrates are known to produce less homogeneous polymers [3], and these should also be tested in P. putida LS46 to broadly conclude whether or not OTR has an effect on monomer composition.
Fig. 3. Final cellular mcl-PHA content (% CDM) at indicated oxygen transfer rate. Error bars indicate the standard deviations between a minimum of two biological replicates. Since kLa measurements more adequately describe conditions below the detection limit of the bioreactor probe and DO measurements more adequately describe high-intensity mixing conditions where kLa may not be reliable due to the response time of the probe (kLa > 310 hour−1), the x-axis is labelled with both parameters.
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Fig. 4. Maximum volumetric and specific productivities of mcl-PHA at indicated oxygen transfer rate. Error bars indicate the standard deviations between a minimum of two biological replicates.
Fig. 5. Distribution of carbon flux at indicated oxygen transfer rates. Values measured after 6 h of cultivation or when DO had reached its intended steady-state value. Error bars indicate the standard deviations between a minimum of two biological replicates. Table 3 Yield reported in other studies for mcl-PHA production from medium chain length fatty acids using P. putida grown in various cultivation strategies. Vprod − volumetric productivity, YPHA/C − yield, mass of mcl-PHA synthesized per unit mass of carbon source utilized. Conditions
N-limited fed batch using octanoic acid N-limited fed batch using octanoic acid N-limited fed batch using octanoate N-limited chemostat using octanoate (D = 0.1 h−1) C-limited fed batch with decanoate C-limited fed batch with nonanoic acid C-limited chemostat with nonanoic acid C-limited fed batch with nonanoic acid
Total Biomass
PHA Content
VProd
YPHA/C
g L−1 CDM
% CDM
g L−1 h−1
g−1
53.0 51.5 8 – 75 71 5.8 71.4
55.4 35.8 4 56.1 74 56 51.7 75.5
0.76 0.41 0.07 – 1.16 1.4 0.7 1.8
0.41 0.21 0.3 0.25 0.86 0.66 0.61 0.78
6
Ref
[48] [49] [50] [51] [10] [12] [52] [53]
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3.4. PHA productivity and yield
accounted for in PHA biomass, residual biomass, and CO2.
Although a low DO environment was shown to cause P. putida LS46 to accumulate PHA and improve final cellular PHA content, this is of little relevance unless the production rates and yield are taken into account. Fig. 4 shows both the volumetric (g L−1 h−1) and specific (mg PHA g residual biomass−1 h−1) productivities obtained under various oxygen transfer rates (OTRs). The specific productivity data (Fig. 4) shows that lower OTRs improved the rate of polymer synthesis from octanoic acid. The highest specific productivity was 227.9 ± 9.8 mg PHA g residual biomass −1 h−1 and was observed when the DO was below detectable limits (kLa = 38 h−1). Further lowering the OTR by reducing aeration to 1 LPM (kLa = 12 h−1) did not negatively affect specific productivity, but reduced the overall volumetric productivity because lower total biomass concentration was obtained. When aeration was replaced with N2 gassing, both growth and PHA synthesis completely ceased (data not shown). This might be expected because initial activation of the fatty acid with coenzyme A consumes ATP, and subsequent oxidation of acyl-CoA to enoyl-CoA (the first suitable intermediate for the PHA synthase) produces FADH2 [38–40]. Consequently, in the absence of oxygen, imbalance of redox couples would rapidly halt growth and PHA production. The maximum specific productivity obtained in this work (227.9 ± 9.8 mg PHA g residual biomass−1 h−1) compares well with the value reported for P. putida KT2442 grown on decanoate under dual nitrogen and carbon limitation, but lower than the value reported for strict nitrogen limitation (430 mg PHA g cells−1 h−1) [46]. Interestingly, the maximum specific productivity observed in low DO conditions was considerably higher than the value observed in several trials of nitrogen-limited batch experiments in which DO was maintained at 40% AS (data not shown). In these experiments, the maximum specific productivity was 140.7 ± 11.0 mg PHA g cells−1 h−1. It appears that a microaerophilic environment may be a stronger driver of PHA synthesis than strict nitrogen limitation in P. putida LS46 when cultured on octanoic acid. Fig. 5 shows that the highest carbon flux to PHA was observed when the DO was maintained below detectable limits, and the resulting yield was 0.62 ± 0.06 g PHA g octanoic acid−1. This was more than a tenfold improvement in yield compared with conditions in which DO was maintained at 10% AS or higher (0.04 ± 0.02 g g−1), and higher than the theoretical yield of PHB from carbohydrate substrates (0.47–0.48 g g−1)[47]. However, the result is still lower than the theoretical maximum yield of PHA from octanoic acid (0.98 g g−1). Table 3 shows yield data reported for mcl-PHA production from various medium chain length fatty acids using P. putida. Yields ranging from 0.21 to 0.41 g g−1 have been reported for N-limited fed batch or chemostat cultivations using octanoate as the carbon source [48–51]. In this work, several N-limited batch trials of P. putida LS46 grown on octanoic acid were performed, and the average yield obtained (0.53 ± 0.06 g g−1) was not significantly different than those observed in low DO conditions with excess ammonium. As shown in Table 3, yields have recently been reported using carbon limitation [10,12,52,53], suggesting that it is possible to further improve yield.
3.6. Applications These results represent, to our knowledge, the first attempt to quantify how growth and mcl-PHA production from fatty acids are modulated in response to non-homogeneous DO environments. Since it was found that low DO environments strongly induce mcl-PHA synthesis from fatty acids, the bioreactor OTR can be adjusted to manipulate the process to maximize productivity without the need to manage C/N ratios. Many high-productivity mcl-PHA cultivation systems have been operated fed-batch mode using structurally related carbon sources like fatty acids. These studies have used various feeding strategies to achieve high-cell density cultures, before limiting carbon [7,12], phosphorus [11], or nitrogen [48]. An oxygen-limited fed batch strategy could be employed in which the DO is maintained as high as possible until the oxygen demand of the culture depletes DO to 1 to 5% AS or lower. This would divert carbon flux into PHA synthesis at a high rate. In a continuous system, it may be possible to use DO content to tune the balance between growth and mcl-PHA synthesis to optimize productivity, without the need for a secondary PHA accumulation reactor. The maximum specific productivity observed in this work using oxygen limitation indicates that, for a PHA accumulation step of a fed batch or multi-stage continuous production system, the residence time required could be significantly reduced using oxygen limitation compared to strict nitrogen limitation. Currently, mcl-PHA production under microaerophilic conditions has not been considered in detail, and could be a platform for improved rates and simultaneous cost reduction, especially at increased bioreactor scale. 4. Conclusions The effect of microaerophilic environments have not been adequately addressed in mcl-PHA production and these conditions are usually blamed for termination of high cell density cultivation processes. These results show for the first time that microaerophilic environments strongly induce mcl-PHA synthesis from fatty acids and the oxygen transfer rate can be manipulated to achieve higher productivity during the PHA accumulation phase than nitrogen limitation. This provides valuable insight for bioreactor operation strategies in scaled-up mcl-PHA production processes, and suggests that the process responds well to inevitable oxygen mass transfer limitations that occur in high cell density cultures and large-scale cultivations. Acknowledgements This work was funded by: 1) Genome Canada, through the Applied Genomics Research in Bioproducts or Crops (ABC) program for the grant titled, “Microbial Genomics for Biofuels and Co-Products from Biorefining Processes”; 2) the government of the Province of Manitoba through the Manitoba Research Innovation Fund (MRIF) and the Manitoba Rural Adaptation Council (MRAC); 3) the Natural Sciences and Engineering Research Council (NSERC); and 4) the University of Manitoba Sir Gordon Wu Graduate Student Scholarship. The authors disclose that no conflict of interest exists between them.
3.5. Carbon dioxide and carbon balance
References
Since CO2 measurements with the MIMS were not available in every experiment, results were back calculated by rearranging Eq. (1). The efficacy of the back-calculated results was verified in experiments where CO2 was measured with the MIMS and was found to be in good agreement. Fig. 5 shows the distribution of consumed carbon at the indicated conditions. The main trade-off in carbon flux was between mass of PHA and residual biomass as oxygen became increasingly limited. Carbon balances (Eq. (1)) for experiments where CO2 was measured ranged from 0.94 to 1.07. This indicates that, within experimental error, the majority of the consumed carbon can be
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Microaerophilic environments improve the productivity of medium chain length polyhydroxyalkanoate biosynthesis from fatty acids in Pseudomonas putida LS46 ⁎
Warren Blunta, , Christopher Dartiailha, Richard Sparlingb, Daniel Gapesc, David B. Levina, Nazim Ciceka a b c
Department of Biosystems Engineering, University of Manitoba, Winnipeg, Manitoba, R3T 5V6, Canada Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada Scion Research, Te Papa Tipu Innovation Park, 49 Sala Street, Private Bag 3020, Rotorua, 3046, New Zealand
A R T I C L E I N F O
A B S T R A C T
Keywords: Polyhydroxyalkanoates Pseudomonas putida Dissolved oxygen Bioreactor Biopolymer Microaerophilic
Medium chain length polyhydroxyalkanoate (mcl-PHA, a commercially desirable biopolymer) synthesis from fatty acids is an oxidative process. Growth and mcl-PHA synthesis were studied in Pseudomonas putida LS46 when subjected to a spectrum of oxygen transfer rates, which were intended to simulate dissolved oxygen (DO) gradients likely to be encountered during process scale-up. It was found that maintaining DO at 1–5% (of air saturation at 30 °C) initiated significant mcl-PHA synthesis and concurrent growth at < 0.5 μmax was also observed. Further reductions in DO resulted in nearly negligible growth, but improved biopolymer synthesis. When the volumetric oxygen mass transfer coefficient (kLa) was reduced to 38 h−1, DO was below detectable limits and both the biopolymer content (57.3 ± 3.8% CDM) and yield (0.62 ± 0.06 g mcl-PHA g octanoic acid−1) reached the maximum observed values. Under these conditions, the maximum rate of polymer synthesis was also observed (227.9 ± 9.8 mg PHA g residual cell mass−1 h−1), and this value was higher than that which could be achieved using nitrogen limitation. Further reductions in oxygen transfer rate did not improve productivity, and PHA synthesis completely ceased when aeration was replaced with N2 gassing. These results suggest that manipulation of the bioreactor oxygen transfer rate during the PHA accumulation phase could be a strategy used to improve overall productivity during a fed batch or continuous feed process.
1. Introduction Medium chain length polyhydroxyalkanoates (mcl-PHAs) are C6-C14 polyesters synthesized by bacteria and have potential value for diverse set of applications [1,2]. Currently production costs are too high for economically viable, commercial scale mcl-PHA production [3]. Development and subsequent scale up of production strategies targeting high overall productivity (a function of reaction rates, yield, and product titer) can help reduce costs and secure the future of mcl-PHA as a commercially valuable biopolymer [4,5]. To synthesize a significant amount of mcl-PHA, most bacteria require nutrient imbalance and to achieve this, medium components like carbon, nitrogen, or phosphate are added in growth-limiting quantities [6]. During mcl-PHA production and many strictly aerobic bioprocesses, gas-to-liquid mass transfer of oxygen is often rate limiting and has a significant impact on the overall productivity [7–9]. Several
studies have supplemented the aeration medium with pure oxygen to increase the driving force for oxygen mass transfer [7,10–12]. Operating bioreactors at elevated pressure has also been used to increase the driving force, although increased pressure has also been shown to have a negative impact on the oxygen mass transfer coefficient, kLa [13,14]. Both strategies are aimed at prolonging the growth phase before oxygen mass transfer becomes rate limiting to achieve higher cell densities and increased volumetric productivity. However, the effect that low dissolved oxygen (DO) environments, likely to be encountered in largerscale and high cell density cultivations, have on mcl-PHA synthesis has not been considered in detail. Low DO conditions have been reported to increase the intracellular scl-PHA content in several microorganisms grown on a variety of substrates [15–17]. Using mixed microbial cultures, Pratt et al. observed increased yield during the early stages of growth in low DO environments, but the rate of accumulation was lower than in high DO environments [18]. Some studies have also
⁎
Corresponding author at: E2-376 Engineering and Information Technology Complex (EITC), 75A Chancellor’s Circle, University of Manitoba, Winnipeg, MB R3T 5V6, Canada. E-mail addresses: [email protected], [email protected] (W. Blunt), [email protected] (C. Dartiailh), [email protected] (R. Sparling), [email protected] (D. Gapes), [email protected] (D.B. Levin), [email protected] (N. Cicek). http://dx.doi.org/10.1016/j.procbio.2017.04.028 Received 20 September 2016; Received in revised form 5 January 2017; Accepted 9 April 2017 1359-5113/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Blunt, W., Process Biochemistry (2017), http://dx.doi.org/10.1016/j.procbio.2017.04.028
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reported changes in the monomeric composition of both scl-and mclPHA when the culture was subject to low DO environments [19,20]. Given that the cellular redox state has been linked to mcl-PHA synthesis [21], oxygen availability and the redox state of the substrate could play a role in mcl-PHA accumulation. Structurally related substrates (alkanes, alkanoic acids) are highly reduced and are preferred for mcl-PHA synthesis due to the high yield [7,10,12]. Production of mcl-PHA from such substrates requires intermediates of the βoxidation cycle [22]. This is an oxidative process (in contrast to synthesis of PHB from glucose) in which FAD and/or NAD+ are used as electron acceptors, and these must be regenerated by electron transport to O2. Therefore, mcl-PHA synthesis from related (highly reduced) substrates under oxygen-limited conditions would be a phenomenon of interest. Little information is available regarding how mcl-PHA production (from fatty acids or other substrates) responds to low DO environments, particularly compared to short chain length (scl) PHA production. Several studies have reported improved scl-PHA polymer production from carbohydrate substrates in low DO environments [15–17]. In this work, we have used a scaled-down approach to assess mclPHA production rates in low DO environments using Pseudomonas putida LS46 grown on octanoic acid in bench scale bioreactors operated in batch mode. The objective of this study was to understand how oxygen mass transfer limitations affect growth and mcl-PHA biosynthesis from fatty acids.
Table 1 Summary of various conditions tested, with DO expressed as a percent of air saturation (AS), and as concentration (mg L−1 and μM) for comparability. Reactor operating parameters required to maintain these conditions, including airflow rate, agitation rates, and resulting volumetric oxygen mass transfer coefficient, are also shown. A minimum of two biological replicates was performed for each condition. Dissolved oxygen content
Vol. O2 Mass Transfer Coefficient, kLa
Air Flow Rate
Mixing Cascade
% AS (30 °C)
mg L−1 O2
μM O2
hour−1
LPM
rpm
0 0 1 5 10 40
0.02a 0.04a 0.07b 0.34b 0.68b 2.71b
0.53 1.28 2.12 10.58 21.16 84.65
38 78 78 78 78 78
3 6 6 6 6 6
250 250 250–410 250–620 250–740 250–900
< < < <
kLa kLa kLa kLa
< < < <
145 310 540c 1016c
a
Reported results were measured with a MIMS. Results measured with electrochemical DO probe. kLa values > 310 h−1 could not be reliably measured due to insufficient response time of probe. Values > 310 h−1 were estimated by extrapolating the trend from the range that could be reliably measured with the probe. b c
flow rate was held constant in all experiments at either three liters per minute (LPM), equal to one volume air per liquid volume per minute (VVM), or six LPM (2 VVM). A mixing cascade (250–900 rpm) was used to maintain DO at levels above detectable limits (non-zero readings with the DO electrode). Since some of the conditions tested were below the threshold of detection of the DO electrode, these were characterized by the oxygen mass transfer coefficient (kLa), which was measured via the dynamic out-gassing method. It was assumed that the probe response could be modeled as first order [25]. Due to insufficient response time of the DO electrode, kLa could not be reliably measured for kLa > 310 h−1, which corresponded to mixing rates of approximately 600 rpm. An HPR-40 dissolved species membrane-inlet mass spectrometer (MIMS) equipped with a HAL 201 RC quadrupole mass analyzer and Faraday Cup detector (Hiden Analytical, Warrington, United Kingdom) was also used to estimate DO concentrations for conditions below the detection threshold of the DO electrode. A summary of the oxygen transfer conditions tested is presented in Table 1. It was assumed that the oxygen solubility in the culture medium at 30 °C was 6.77 mg L−1, which was measured previously in a minimal medium [26].
2. Materials and methods 2.1. Microorganism, medium, and substrates The production strain used in this study was Pseudomonas putida LS46 [23]. Glycerol stock cultures were stored at −80 °C, and were periodically revived and maintained on agar plates stored at 4 °C. The medium used was Ramsay’s medium [24], with 20 mM octanoic acid added as a carbon source. Baffled flasks (500 mL flask with 100 mL working volume) were used for the reactor inoculum and were incubated at 30 °C and 150 rpm shaking. The initial pH of the medium in flask cultures was adjusted to 7.0 with the addition of NaOH. The medium for bioreactor experiments was slightly modified by increasing the concentration of (NH4)2SO4 to 11.35 mM (1.5 g L−1) to ensure excess ammonium throughout each trial. 2.2. Reactor setup and preparation All reactor experiments were conducted in a 7 L glass, round-bottom reactor with a 3 L working volume. The reactor was equipped with a Rushton impeller, three baffles, a polarographic DO electrode, and a pH electrode. All reactor equipment was purchased from Applikon Biotechnology (Foster City, CA). The oxygen transfer capability of the reactor was improved through the addition of two Pyrex microbubblers, which were connected to the existing sparging tube with Masterflex LS25 tubing (4.8 mm inner diameter), and located underneath the impeller. The medium, substrate, and 0.2 mL of a silicone antifoaming agent (obtained from Husky Energy in Minnedosa, MB) were added to the reactor prior to autoclaving at 121 °C for 40 min. After cooling, the reactor was stabilized with air overnight to allow polarization of the DO electrode, and maintained at 30 °C. Prior to addition of the inoculum, a one-point calibration of the DO electrode was performed at operating air flow rate, and this condition was assigned a value of 100% of air saturation (AS). The pH was adjusted to a set point of 6.5 via addition of 1 M HCl and 1 M NaOH through automated peristaltic pumps.
2.4. Reactor monitoring Experiments were initiated with the addition of 1% (vol−1) inoculum. At this time the medium was saturated with air, (DO = 100%) and the DO was allowed to fall until the indicated set point was reached. A 40 mL sample of culture was taken in 1–2 h intervals from the reactor for analysis of biomass, PHA, residual octanoic acid, and residual free ammonia. These procedures are described below.
2.5. Measurement techniques 2.5.1. Total biomass and residual (PHA-free) biomass Each sample from the reactor was transferred into pre-weighed 50 mL centrifuge tubes, and the total biomass was immediately separated from the supernatant by centrifugation (4000g for 20 min at 4 °C). The supernatant was stored at −20 °C. The pellet was then oven dried at 60 °C until no further loss in mass was detected (24 h), and then total biomass was determined gravimetrically. Residual (PHAfree) biomass was estimated as the difference between total biomass and the mass of detected PHA monomers.
2.3. Oxygen transfer conditions Air was used for sparging the culture in all studies. The volumetric 2
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cells had grown to an average cell density of 0.7 g L−1 CDM. This portion of the growth curve (after 6 h) was used to calculate the specific growth rates. Fig. 2 shows the change in specific growth rate at different DO conditions. Due to the lack of sensitivity of the bioreactor probe in periods when the DO was not controlled (constant mixing at 250 rpm and constant aeration at either 3 LPM or 6 LPM), the MIMS was used to confirm the DO concentration in these two conditions (Table 1). The relationship between growth rate and DO concentration followed Monod kinetics (R2 = 0.99) and the fitted parameters indicated μmax = 0.57 h−1 and the oxygen half saturation constant (Ks) to be 0.19 mg L−1 (2.7% AS at 30 °C). This Ks value is relatively low compared to the values reported for other Pseudomonas species and strains and indicates a high affinity for oxygen [30,31]. A possible explanation for this may be the presence of two putative subunits for a cytochrome bd complex in the genome annotation for the respiratory chains in P. putida LS46 (www.img.jgi.doe.gov). Cytochrome bd is a terminal oxidoreductase with a very high affinity for oxygen (but lower ATP yield) and is expressed in several microorganisms in stressful environments, including microaerophilic conditions [32,33]. Tseng et al. [34] have shown that cytochrome bd expression increases dramatically when DO falls below 10% AS, and reached a maximum at 0% AS in E. coli cultures. Based on the relatively steep reduction in growth rate at DO < 10% AS in P. putida LS46, it may be possible that this also occurs in LS46. Proteomic analysis would be necessary to confirm this. Overall, the data indicates that P. putida LS46 is capable of not only survival, but also growth in low DO environments.
2.5.2. mcl-PHA and fatty acid analysis Preparation and analysis of the dried biomass samples for cellular mcl-PHA content followed the acid-catalyzed methanolysis procedure previously described [3]. The same procedure was followed for analysis of octanoic acid in the culture medium, except that 1 mL of thawed culture supernatant (allowed to come to room temperature and vortexed thoroughly) was added to each reaction vial. Determination of mcl-PHA content of the cells and octanoic acid was accomplished using an Agilent 7890A gas chromatograph equipped with a splitsplitless inlet (operated in split mode, split ratio 10:1), a DB23 capillary column (Agilent, 30 m × 250 μm × 0.25 μm), and a flame ionization detector. Method operating parameters and peak quantification are described elsewhere [3]. 2.5.3. Residual free ammonium Residual ammonium in the culture medium was determined via the Lachat QuikChem® Method 10–107-06-1-J. Briefly, this method uses a spectrophotometer coupled to a flow injection system (Lachat Instruments, CO) that measures the blue color change resulting from the reaction of alkaline phenol and sodium hypochlorite with ammonia to form indophenol blue, which is measured at 630 nm. 2.6. Productivity and yield calculations Volumetric productivity of a batch was calculated as the maximum value of PHA titer divided by the cultivation time at which it was observed. Specific productivity was calculated as the increase in PHA concentration in a given time divided by the mean residual biomass measured during that time. Yield was calculated as the slope of a regression of PHA titer (g L−1) and substrate uptake (g L−1). The maximum theoretical yield of mcl-PHA from octanoic acid was calculated as 0.98 g g−1 following the approach used by Weusthuis et al. with the assumption that the polymer synthesized from octanoic acid can be treated as a homopolymer of C8 monomers [27].
3.2. PHA accumulation The maximum cellular PHA content data (Fig. 3) shows that little PHA was detected for conditions in which DO was maintained at 10% AS or higher. A significant increase in cellular PHA content (up to 41.4% of cell dry mass, CDM) was consistentlyevident when DO was maintained at l% AS or lower. At the lowest OTR tested (kLa = 38 h−1), the final cellular mcl-PHA content was 57.3% CDM. The final cellular PHA content was somewhat variable (23 to 38% CDM) when the DO was maintained at 5% AS. This variability may suggest this condition could be close to a switching point from growth to PHA synthesis in response to stress from low DO. Due to fluctuations in the PIDcontrolled DO measurement, further resolution was not possible. It appears that little PHA is accumulated when DO > 5% AS and whilst at DO ≤ 5% AS, cells begin to accumulate a significant amount of PHA. On the basis of final PHA content these results indicate that growth in shaker flasks, with the same C/N ratio and low working volumes, is oxygen limited. The relationship between oxygen limitation and PHA accumulation was first established as a way for disposal of excess reducing power during synthesis of PHB from glucose in Azotobacter vinelandii [17]. Since there is no reductive step during synthesis of mcl-PHA from βoxidation intermediates, this cannot explain observation of mcl-PHA synthesis from fatty acids in response to low DO. High NAD(P)H/NAD (P)+ ratios have been linked to mcl-PHA synthesis from fatty acids [21,35], as well as non-related substrates [36]. This redox imbalance would likely occur at low DO and cause inhibition of key enzymes in the TCA cycle [37]. If PHA is produced from intermediates of the βoxidation cycle, conversion of either the enoyl-CoA monomers (via an R-specific enoyl-CoA hydratase, PhaJ) or (S)-3-hydroxyacyl-CoA monomers (via epimerase enzyme) to (R)-3-hydroxyacyl-CoA monomers requires only one oxidative step. In this step, the acyl-CoA dehydrogenase enzyme oxidizes acyl-CoA monomers to enoyl-CoA monomers using FAD as the electron acceptor [1,38–40]. Storage of these monomers as PHA (primarily C8 and C10 in P. putida) would generate fewer reduced electron carriers compared to complete oxidation. Completion of the β-oxidation cycle requires oxidation of 3-hydroxya-
2.7. Carbon dioxide measurement and carbon balancing Carbon dioxide was measured in the reactor headspace at a mass-tocharge ratio of 44 using the MIMS. Further detail on MIMS calibration and quantification of CO2 is given elsewhere [28]. A carbon balance at any time during the fermentation could be performed as shown in Eq. (1).
[6 × C6 + 8 × C8 + 10 × C10] + 4 × (moles residual biomass ) t
+ C Balance =
∫0
[CO2] dt 8 × (moles octanoic acid consumed ) (1)
Where C6, C8, C10 represent moles of the corresponding monomer subunits produced (determined by GC). It was assumed that elemental composition of residual biomass could be approximated by the formula C4H7O2N and as having a molecular mass of 101 g mol−1 [29]. For experiments in which CO2 measurements were not available, it was assumed that the left hand side of this equation was equal to 1, and the equation was rearranged to back-calculate CO2 production. 3. Results and discussion 3.1. Growth rate Fig. 1 shows sample growth curves for an oxygen-excess condition (Fig. 1A) as well as an oxygen-limited condition (Fig. 1B). Specific growth rates were assessed as the increase in residual (PHA-free) biomass once the target DO content was reached. The target DO was typically reached about 6 h after inoculation in each trial, when the 3
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Fig. 1. A) Growth curve for A) condition in which DO was maintained at 40% and B) condition in which DO was below detectable limits with the bioreactor probe (kLa = 38 h−1). Data includes total biomass (○), residual biomass (▯), PHA concentration (☐), and cellular PHA content (♦). The DO content over time is represented by the continuous line. Error bars indicate standard deviations between biological replicates.
cyl-CoA to 3-ketoacyl-CoA, which uses NAD+ as the electron acceptor. Furthermore, subsequent oxidation of acetyl-CoA (produced from each completion of the β-oxidation cycle) via the TCA cycle generates 3 NADH and 1 FADH2 [1,41]. Therefore, due to redox imbalance at low DO the cell may not be able to further metabolize the available acylCoA, instead leaving it accessible to the monomer offering enzymes for PHA synthesis. Thus, mcl-PHA synthesis from fatty acids when oxygen is limited cannot be a ‘sink’ for electrons that has been previously described in PHB synthesis [17], but may be a mechanism for storing electrons as PHA to maintain a balanced redox state. A similar assertion was also made for degradation of PHB to supply reducing power required to elicit an anti-oxidant stress response in P. putida 14-3 [42].
This is not the case for synthesis of scl-PHA or mcl-PHA from carbohydrate substrates, since both pathways include reductive steps. Although improved scl-PHA production from carbohydrate substrates in low DO environments has been reported [15–17], we did not observe any improvement in mcl-PHA production from P. putida LS46 cultured on crude glycerol using sequentially lower surface-area-to volume ratios in baffled shaker flasks (data not shown). Further investigation is required, but this result is in agreement with other studies of mclPHA synthesis from non-related substrates via de novo fatty acid synthesis [43–45].
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Fig. 2. Specific growth rate (μ) at indicated oxygen transfer rate. Values measured after 6 h of cultivation, or when DO had reached its intended steady state value. Error bars indicate the standard deviations between a minimum of two biological replicates. Since kLa measurements more adequately describe conditions below the detection limit of the bioreactor probe and DO measurements more adequately describe high-intensity mixing conditions where kLa may not be reliable due to the response time of the probe (kLa > 310 hour−1), the x-axis is labelled with both parameters.
3.3. Monomer composition of polymer
Table 2 Summary of observed monomer composition among the conditions tested. The monomer composition was measured as the mean of all samples taken after the DO had reached its target concentration (generally after 6 h cultivation). A minimum of two biological replicates was performed for each condition.
The monomer compositions of the polymer synthesized under the conditions tested are shown in Table 2. For conditions in which PHA was accumulated the composition was not significantly different in any case. For these cases, the mean monomer composition was 5.9 ± 0.9% C6, 92.3 ± 1.1% C8, and 1.9 ± 0.6% C10. However, after the exogenous carbon source was depleted, the C8 monomers appeared to be consumed first, causing an enrichment of C6 and C10 monomers and significantly different monomer distributions. This suggests that prolonged carbon limitation may cause inconsistent product quality, and the timing of harvest might be critical. Dissolved oxygen has been previously observed to play a role in altering the monomer composition in both scl-PHA and mcl-PHA production. Lefebvre et al. report that low DO (1–4% AS) caused more than a two-fold increase in yield of poly-(3-hydroxyvalerate) from propionate during production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose and propionate using C. necator as compared to conditions when the DO was maintained at 50–70% AS [20]. Fernández et al. report that during mcl-PHA production from crude oleic acid using P. aeruginosa, lowering the OTR caused significant increase of C8, C12:1 and the appearance of C14:2 monomers, while C10, C12, and C14:1 all decreased [19]. The major distinction between these two previous
Dissolved oxygen content
kLa
Monomer Composition (mol%)
% AS (30 °C)
hour−1
C6
0 (3 LPM air) 0 (6 LPM air) 1 5 10 40
38 78 78 78 78 78
6.0 6.0 5.6 4.8 6.1 6.3
< < < <
kLa kLa kLa kLa
< < < <
145 310 540 1016
C8 ± ± ± ± ± ±
1.1 0.4 1.2 0.4 1.2 0.3
92.5 91.9 92.4 93.2 92.3 91.6
C10 ± ± ± ± ± ±
0.8 0.5 2.1 0.1 0.8 0.4
1.4 2.0 1.6 2.0 1.6 2.8
± ± ± ± ± ±
0.4 0.6 0.3 0.3 0.4 0.9
studies and this work is that we observed lower OTRs to significantly improve biopolymer content and productivity. Different substrates are known to produce less homogeneous polymers [3], and these should also be tested in P. putida LS46 to broadly conclude whether or not OTR has an effect on monomer composition.
Fig. 3. Final cellular mcl-PHA content (% CDM) at indicated oxygen transfer rate. Error bars indicate the standard deviations between a minimum of two biological replicates. Since kLa measurements more adequately describe conditions below the detection limit of the bioreactor probe and DO measurements more adequately describe high-intensity mixing conditions where kLa may not be reliable due to the response time of the probe (kLa > 310 hour−1), the x-axis is labelled with both parameters.
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Fig. 4. Maximum volumetric and specific productivities of mcl-PHA at indicated oxygen transfer rate. Error bars indicate the standard deviations between a minimum of two biological replicates.
Fig. 5. Distribution of carbon flux at indicated oxygen transfer rates. Values measured after 6 h of cultivation or when DO had reached its intended steady-state value. Error bars indicate the standard deviations between a minimum of two biological replicates. Table 3 Yield reported in other studies for mcl-PHA production from medium chain length fatty acids using P. putida grown in various cultivation strategies. Vprod − volumetric productivity, YPHA/C − yield, mass of mcl-PHA synthesized per unit mass of carbon source utilized. Conditions
N-limited fed batch using octanoic acid N-limited fed batch using octanoic acid N-limited fed batch using octanoate N-limited chemostat using octanoate (D = 0.1 h−1) C-limited fed batch with decanoate C-limited fed batch with nonanoic acid C-limited chemostat with nonanoic acid C-limited fed batch with nonanoic acid
Total Biomass
PHA Content
VProd
YPHA/C
g L−1 CDM
% CDM
g L−1 h−1
g−1
53.0 51.5 8 – 75 71 5.8 71.4
55.4 35.8 4 56.1 74 56 51.7 75.5
0.76 0.41 0.07 – 1.16 1.4 0.7 1.8
0.41 0.21 0.3 0.25 0.86 0.66 0.61 0.78
6
Ref
[48] [49] [50] [51] [10] [12] [52] [53]
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3.4. PHA productivity and yield
accounted for in PHA biomass, residual biomass, and CO2.
Although a low DO environment was shown to cause P. putida LS46 to accumulate PHA and improve final cellular PHA content, this is of little relevance unless the production rates and yield are taken into account. Fig. 4 shows both the volumetric (g L−1 h−1) and specific (mg PHA g residual biomass−1 h−1) productivities obtained under various oxygen transfer rates (OTRs). The specific productivity data (Fig. 4) shows that lower OTRs improved the rate of polymer synthesis from octanoic acid. The highest specific productivity was 227.9 ± 9.8 mg PHA g residual biomass −1 h−1 and was observed when the DO was below detectable limits (kLa = 38 h−1). Further lowering the OTR by reducing aeration to 1 LPM (kLa = 12 h−1) did not negatively affect specific productivity, but reduced the overall volumetric productivity because lower total biomass concentration was obtained. When aeration was replaced with N2 gassing, both growth and PHA synthesis completely ceased (data not shown). This might be expected because initial activation of the fatty acid with coenzyme A consumes ATP, and subsequent oxidation of acyl-CoA to enoyl-CoA (the first suitable intermediate for the PHA synthase) produces FADH2 [38–40]. Consequently, in the absence of oxygen, imbalance of redox couples would rapidly halt growth and PHA production. The maximum specific productivity obtained in this work (227.9 ± 9.8 mg PHA g residual biomass−1 h−1) compares well with the value reported for P. putida KT2442 grown on decanoate under dual nitrogen and carbon limitation, but lower than the value reported for strict nitrogen limitation (430 mg PHA g cells−1 h−1) [46]. Interestingly, the maximum specific productivity observed in low DO conditions was considerably higher than the value observed in several trials of nitrogen-limited batch experiments in which DO was maintained at 40% AS (data not shown). In these experiments, the maximum specific productivity was 140.7 ± 11.0 mg PHA g cells−1 h−1. It appears that a microaerophilic environment may be a stronger driver of PHA synthesis than strict nitrogen limitation in P. putida LS46 when cultured on octanoic acid. Fig. 5 shows that the highest carbon flux to PHA was observed when the DO was maintained below detectable limits, and the resulting yield was 0.62 ± 0.06 g PHA g octanoic acid−1. This was more than a tenfold improvement in yield compared with conditions in which DO was maintained at 10% AS or higher (0.04 ± 0.02 g g−1), and higher than the theoretical yield of PHB from carbohydrate substrates (0.47–0.48 g g−1)[47]. However, the result is still lower than the theoretical maximum yield of PHA from octanoic acid (0.98 g g−1). Table 3 shows yield data reported for mcl-PHA production from various medium chain length fatty acids using P. putida. Yields ranging from 0.21 to 0.41 g g−1 have been reported for N-limited fed batch or chemostat cultivations using octanoate as the carbon source [48–51]. In this work, several N-limited batch trials of P. putida LS46 grown on octanoic acid were performed, and the average yield obtained (0.53 ± 0.06 g g−1) was not significantly different than those observed in low DO conditions with excess ammonium. As shown in Table 3, yields have recently been reported using carbon limitation [10,12,52,53], suggesting that it is possible to further improve yield.
3.6. Applications These results represent, to our knowledge, the first attempt to quantify how growth and mcl-PHA production from fatty acids are modulated in response to non-homogeneous DO environments. Since it was found that low DO environments strongly induce mcl-PHA synthesis from fatty acids, the bioreactor OTR can be adjusted to manipulate the process to maximize productivity without the need to manage C/N ratios. Many high-productivity mcl-PHA cultivation systems have been operated fed-batch mode using structurally related carbon sources like fatty acids. These studies have used various feeding strategies to achieve high-cell density cultures, before limiting carbon [7,12], phosphorus [11], or nitrogen [48]. An oxygen-limited fed batch strategy could be employed in which the DO is maintained as high as possible until the oxygen demand of the culture depletes DO to 1 to 5% AS or lower. This would divert carbon flux into PHA synthesis at a high rate. In a continuous system, it may be possible to use DO content to tune the balance between growth and mcl-PHA synthesis to optimize productivity, without the need for a secondary PHA accumulation reactor. The maximum specific productivity observed in this work using oxygen limitation indicates that, for a PHA accumulation step of a fed batch or multi-stage continuous production system, the residence time required could be significantly reduced using oxygen limitation compared to strict nitrogen limitation. Currently, mcl-PHA production under microaerophilic conditions has not been considered in detail, and could be a platform for improved rates and simultaneous cost reduction, especially at increased bioreactor scale. 4. Conclusions The effect of microaerophilic environments have not been adequately addressed in mcl-PHA production and these conditions are usually blamed for termination of high cell density cultivation processes. These results show for the first time that microaerophilic environments strongly induce mcl-PHA synthesis from fatty acids and the oxygen transfer rate can be manipulated to achieve higher productivity during the PHA accumulation phase than nitrogen limitation. This provides valuable insight for bioreactor operation strategies in scaled-up mcl-PHA production processes, and suggests that the process responds well to inevitable oxygen mass transfer limitations that occur in high cell density cultures and large-scale cultivations. Acknowledgements This work was funded by: 1) Genome Canada, through the Applied Genomics Research in Bioproducts or Crops (ABC) program for the grant titled, “Microbial Genomics for Biofuels and Co-Products from Biorefining Processes”; 2) the government of the Province of Manitoba through the Manitoba Research Innovation Fund (MRIF) and the Manitoba Rural Adaptation Council (MRAC); 3) the Natural Sciences and Engineering Research Council (NSERC); and 4) the University of Manitoba Sir Gordon Wu Graduate Student Scholarship. The authors disclose that no conflict of interest exists between them.
3.5. Carbon dioxide and carbon balance
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Since CO2 measurements with the MIMS were not available in every experiment, results were back calculated by rearranging Eq. (1). The efficacy of the back-calculated results was verified in experiments where CO2 was measured with the MIMS and was found to be in good agreement. Fig. 5 shows the distribution of consumed carbon at the indicated conditions. The main trade-off in carbon flux was between mass of PHA and residual biomass as oxygen became increasingly limited. Carbon balances (Eq. (1)) for experiments where CO2 was measured ranged from 0.94 to 1.07. This indicates that, within experimental error, the majority of the consumed carbon can be
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