Dynamic accumulation and degradation of poly(3-hydroxyalkanoate)s in living cells of Azotobacter vinelandii UWD characterized by ¹³C NMR

Abstract

The synthesis and degradation of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) by Azotobacter vinelandii UWD were investigated using natural abundance solution ¹³C nuclear magnetic resonance (NMR) in vivo in shake flask culture and in fermenter culture. The synthesis and the degradation of poly(3-hydroxyalkanoate)s (PHA) monomers hydroxybutyrate (HB) and hydroxyvalerate (HV) had different rates. The amount of HB and HV increased dramatically in the initial degradation stage. The results suggest that the intracellular PHA of strain UWD was the subject of dynamic metabolic processing. ¹³C NMR in vivo analysis provided a rapid, easy, accurate, non-destructive method to obtain valuable information on the metabolism of PHA.

¹³C nuclear magnetic resonance spectroscopy in vivo, Bacterial polyester, Intracellular poly(3-hydroxyalkanoate)s accumulation, Degradation

1 Introduction

Poly(3-hydroxyalkanoate)s (PHA) are produced by a wide range of bacteria as carbon and energy reserve polymers when carbon sources are in excess and other nutrients, such as nitrogen, phosphate, oxygen, or sulfur become limited [1–3]. They are considered as an ideal storage material because they are highly reduced and water insoluble; therefore, no osmotic pressure effects will be induced inside the cell. PHA are also a family of biodegradable and biocompatible polymers having many interesting properties, such as piezoelectricity and nonlinear optical activity, which may offer some high-value applications [2].

Since the 1980s, nuclear magnetic resonance (NMR) technology has been used to investigate various aspects of PHA including the monomer compositions [4–6], cellular content [7,8], conformational analysis [8,9], monomer linkage sequence [10], copolymer analysis [11,12] and PHA metabolic pathways studies [13,14]. In 1986, Jacob and his colleagues observed four resonance peaks when they first reported the direct measurement of PHB in whole cells using solid-state ¹³C NMR [15]. ¹³C NMR spectroscopy is a valuable non-destructive method for monitoring polymer formation and degradation and has the advantages of accuracy, speed and sensitivity [16]. ¹³C labelling technology can be used to monitor the metabolic pathways of tagged substrates to obtain novel information on cell metabolism that can not be obtained using traditional methods. Thus, NMR has been used widely in studying the physical and chemical properties and the metabolism of PHA in intact cells [14,17–19].

However, until recently, in vivo NMR technology for measuring such PHA properties has not been fully developed. Here we present a study of PHA metabolism in living cells using NMR technology with the strain Azotobacter vinelandii UWD, to investigate the process of PHB and poly(3-hydroxybutyrate-co-hydroxyvalerate) P(HB-co-HV) accumulation and degradation in vivo. In P(HB-co-HV) degradation studies, time-lapse samples taken at different culture times were also compared. This study could lead to deeper understanding of PHA metabolism in cells and gives a good example of metabolic research of PHA in living cells using NMR technology.

2 Materials and methods

2.1 Preparation of bacterial cells

A. vinelandii UWD, a gift from Dr. W.J. Page of University of Alberta (Alta., Canada), was grown in mineral salts medium (g l⁻¹): Na₂HPO₄ 3.8, KH₂PO₄ 2.65, (NH₄)₂SO₄ 0.5, MgSO₄ 0.2, 1 ml l⁻¹ trace elements (0.218 g CoCl₂, 9.79 g FeCl₃, 7.89 g CaCl₂, 0.118 g NiCl₃, 0.105 g CrCl₃·6H₂O, 0.156 g CuSO₄·5H₂O were dissolved in 1 l 1 M HCl). The cells were grown on two types of carbon substrates: (1) 20 g l⁻¹ sucrose (for PHB accumulation) and (2) 20 g l⁻¹ sucrose mixed with 3 ml l⁻¹ sodium valerate (for P(HB-co-HV) accumulation). In the shake flask experiments, 10 ml overnight culture was sub-cultured in 400 ml medium in a 1-l shake flask and incubated at 37°C with shaking at 200 rpm. In the fermenter experiments, 3 l of culture were incubated in a 5-l fermenter (Shimoden model P-115-V) at 37°C with a stirring speed of 350 rpm and airflow rate of 5 l min⁻¹. In the degradation experiments, 400 ml of 2–3-day cultures of bacteria were centrifuged under sterile conditions before transfer into 400 ml mineral salts medium (without carbon sources) in a 1-l shake flask. This was then cultured at 37°C with shaking at 200 rpm.

10 ml of the flask culture and 2 ml of fermenter culture were collected for NMR experiments at different times. The samples were centrifuged at 4°C and 3000×g for 15 min. The cell pellets were washed with 4 ml distilled water and the centrifugation step was repeated. Cells were stored at 4°C before the NMR measurements.

2.2 NMR analysis

Cells were resuspended in 25% deuterium oxide (D₂O), and then 0.5 ml of the suspension was transferred into a 5-mm NMR tube. The ¹³C NMR spectra were obtained at 125.759 MHz using a Bruker AM 500 spectrometer. The experiments were carried out at 310 K (37°C) to achieve better signal-to-noise ratios (S/N) and sharper peaks. Spectra of the PHB samples were collected with 512 scans and spectra of the P(HB-co-HV) samples were collected with 1024 scans. The parameters used for the NMR spectra acquisition were: line broadening of 10 Hz, pulse width of 4.5 μs (90° pulse), delay between pulses of 5 s and data block of 32 K, using a spectra width of 29412 Hz. The degradation and accumulation curves were evaluated by measuring the integral areas of the CH₃ resonance of the HB and HV monomers.

3 Results and discussion

3.1 In vivo evaluation of PHB synthesis and degradation

The degradation and accumulation curves of PHB in strain UWD grown in shake flask are presented in Fig. 1. The rate of PHB degradation was high until about 85% of the original PHB had been degraded, after which the rate decreased (Fig. 1a). The different degradation rates may be caused by the different concentrations of intracellular PHB. Additionally, PHB is not only a conserved carbon source but is also a constitutive cell component, which can not be completely degraded. The results also showed that the synthesis curve of PHB (Fig. 1b) was markedly different from the degradation curves. The PHB accumulation reached a maximum within 40 h and then began to degrade, which probably reflected the exhaustion of an available carbon source [20].

1

Time courses of PHB degradation (a) and accumulation (b) monitored by ¹³C NMR in vivo during the shake flask cultivation of A. vinelandii UWD in a mineral medium containing sucrose.

3.2 In vivo evaluation of P(HB-co-HV) accumulation

Sucrose mixed with sodium valerate was used as the carbon source for strain UWD to synthesize P(HB-co-HV). Typical spectra for the polymer accumulation in shake flask are presented in Fig. 2 with the corresponding accumulation curves shown in Fig. 3. Since valerate had a negative effect on polymer formation [21], the accumulation of HB monomers with valerate was notably slower than when only sucrose was used as the carbon source (Fig. 1b). Since the valerate concentration was initially high, HV was synthesized more rapidly than HB with an exponential rate of accumulation. After cultivation for 20 h, the amount of HV reached 66% of its maximum and its accumulation rate began to slow (Fig. 3a). HV amount reached its maximum after 52 h and then began to decrease. However, HB began to accumulate exponentially after 40 h. During the first 24 h, the ratio of HV to HB increased continuously (Fig. 3b), which indicated that HV was the main product of the accumulation and that the bacterial enzyme system preferentially utilized valerate as the substrate. After this time, this ratio began to fall, which suggested that the bacteria mainly synthesize the HB monomer rather than HV in the cell. These trends suggest that the cells could degrade excessive valerate to ameliorate the environment. When valerate was nearly consumed, its negative effect on the polymer formation was eliminated and the cells more quickly produced the polymer [21]. This phenomenon also showed the ability of strain UWD to acclimate to the environment.

2

Natural abundance solution ¹³C NMR spectra of P(HB-co-HV) accumulation by strain A. vinelandii UWD cultured in shake flask.

3

Time courses of P(HB-co-HV) accumulation monitored by ¹³C NMR in vivo during the shake flask cultivation of A. vinelandii UWD in a mineral medium containing sucrose mixed with sodium valerate: (a) accumulation of HA monomers; (b) V5 to B4 ratio.

In the fermenter culture, as in the flask experiments, the HV content reached a peak then remained constant (data not shown). However, the HB monomer began to increase rapidly after cultivation for 22 h. The ratio of HV to HB changed in the fermenter culture in the same way as seen in the flask culture, but the PHA accumulation rate was much faster. The maximum value of the HV to HB ratio was about 0.85:1 after 20 h in the fermenter culture while the maximum value was about 1.5:1 after 22 h in the shake flask culture. During the bacterial growth lag phase, the ratio of HV to HB remained at about 1:50 in the fermenter.

The polymer synthesized by strain UWD cultured in the presence of sucrose and sodium valerate was a type of copolymer [21]. In our experiments the ratio of HV to HB changed with the environment and the extension of PHA accumulation. As seen in Fig. 3a, the HV content decreased after reaching its maximum while the HB content increased exponentially. The results suggest that some of the HV monomers degraded during PHA accumulation. Therefore, the amount of copolymer as an energy and carbon storage did not remain static after it was formed but the polymer was continuously synthesized and degraded as the bacteria grew. The accumulation of P(HB-co-HV) was a dynamic process of synthesis and degradation. This result is consistent with the cyclic nature of PHA metabolism in Alcaligenes eutrophus [20].

3.3 In vivo evaluation of P(HB-co-HV) degradation

Fig. 4 illustrates the degradation of the PHA synthesized in the flask culture (after cultivation for 72 h). The amounts of HB and HV increased dramatically in the initial degradation stage, indicating that the PHA metabolic pathway still actively synthesized PHA [16]. PHA was then degraded to maintain cell metabolism in the absence of a carbon source. The HV to HB ratio increased slowly from 25 to near 40% during the degradation process (Fig. 4b), suggesting that the bacteria preferred to utilize HB rather than HV. Cells cultured for 22 h (sample A, during the initial stage of PHA accumulation) and 42 h (sample B, during the lag stage of PHA accumulation) in the fermenter were also used to evaluate the degradation of PHA. The time courses for the measurements of the HA monomer contents and the HV to HB ratio had the same pattern as those for the flask cultures (data not shown). A similar increase of the HA monomer amount was also observed at the initial stage of degradation. The increase of sample A (about 160%) was a little higher than that of sample B (about 140%), perhaps because the two samples were taken at different periods during the PHA accumulation.

4

Time courses of P(HB-co-HV) rich cell degradation monitored by ¹³C NMR in vivo during the shake flask cultivation of A. vinelandii UWD in a mineral medium: (a) degradation of HA monomers; (b) V5 to B4 ratio.

In the P(HB-co-HV) degradation studies, it is clear that even at the beginning, when the cells cultured in either the shake flask or the fermenter were suspended in a carbon-free medium, the polymer formation had not reached a maximum. This possibility was also observed in Pseudomonas oleovorans [16] and may be an important consideration for strain UWD fermentation. In conclusion, the intracellular PHA of strain UWD underwent dynamic metabolic processing during both PHA accumulation and degradation.

Acknowledgements

The authors gratefully acknowledge the partial financial support provided by THSJZ of Tsinghua University. The bacteria used in this experiment were provided by Dr. W.J. Page of University of Alberta (Canada).