P-27 海洋性紅色光合成細菌によるバイオポリエステル生産システムの開発
樋口 美栄子
理化学研究所 環境資源科学研究センター バイオ高分子研究チーム

Advantages of photosynthetic bacteria➢ Anoxygenic photosynthetic bacteria especially purple nonsulfur bacteria are known to produce high PHA content and shown to have nitrogen fixation ability as well as
CO2 fixation ability.
➢ The majority of purple nonsulfur bacteria can grow as photoautotrophs or photoheterotrophs in the light, and some strains can grow aerobically in the dark as chemoheterotrophs.
This variety of growth style can expand the possibilities for wide range of application.
➢ We demonstrated that marine purple photosynthetic bacteria produced high molecular weight of polyhydroxyalkanoate (PHA) which is useful property for industrial polymer
production and produced PHA using artificial seawater as a culture medium (Higuchi-Takeuchi M et al. 2016).
◆ Anoxygenic photosynthetic bacteria
Growth condition
Oxygen-limited or anaerobic
conditions
Purple non-sulfur Organic compounds, Facultative anaerobic (canH2
grow with or without oxygen)
H2S, H2
H2S,
Organic compounds
Organic compounds

CO2 (Carbon)
N2 (Nitrogen)

500 nm

500 nmPhotosynthesis

400

Nitrogen fixation

Biodegradation

350
300
250200

Nutrient rich (Growth)
Nitrogen-limited,
0.5% acetate

150100

Seawater(Minerals)

500Ros.
visakhapatna
mensisRos. goensis

Ros. marina

Rdv.
visakhapatnamense

SulfurRdv.
tesquicola

Rdv.
sulfidophilum

Rdv. imhoffii

Rdv.
euryhalinum

Rps. marina

Mch.
bheemlicum

Tpc.
mangrovi

Obligatory anaerobic
Facultative anaerobic,
Weak CO2 fixation activity
Aerobic,
No Rubisco activity

Thc. marina

Green sulfur
Green non-sulfur(filamentous)
Aerobic
photosynthetic◆ Marine purple photosynthetic bacteria factory

Rdv. visakhapatnamense(Purple non-sulfur)

Mch. bheemlicum(Purple sulfur)

Electron donor
H2S, H2

PHA content (mg/L culture)

Species
Purple sulfur◆ PHA production by marine purple photosynthetic bacteria

BiPolymer

Non-sulfur

Effect of growth light conditions➢ PHA production and cell growth under different wavelengths of far-red light➢ Relationship between PHA production and cell growth under
different light intensities• Bacterial cells grow well up to 4 days of cultivation under illumination with 800 nm and 850 nm LED compared to 730 nm
LED. Whereas 730 nm LED lighting was the best for bacterial growth after 5 days of cultivation. These imply that shorter
wavelength of LED light (730 nm) might be able to penetrate the cells even at high cell density.

• A linear correlation between PHA contents and dry cell weights (DCWs) were found under
low light conditions (R2 = 0.6289). This indicates that PHA production is dependent on cell
growth under low light conditions. On the other hand, a correlation coefficient between PHA
contents and DCWs under high light conditions were quite low (R2 =0.0028), indicating
there is no linear relationship.

• PHA concentrations (mg/L) increased with increasing incubation time. PHA concentrations showed the highest after 4 days
cultivation (1.2 g/L) at low light of 800 nm LED.
83000

1600

3000

Low light

800 nm246810

2500

2000

2000

730 nmTime (Day)

800 nm

DCW(mg/L)

2500

1000

012Time (days)

1500
1000

y = 41.342x + 43.24
R² = 0.6289

500

850 nmy = -31.113x + 2281.2
R² = 0.1793

500

00020406005PHA content (wt%)

LL: low light (8 w/m2), ML: middle light (20 w/m2), HL: high light (50 w/m2)
Fig. 1. Growth of marine purple nonsulfur bacteria under different lightconditions. Cells were cultured for 1 to 12 days under 8 W/m2 of 730 nm,
800 nm and 850 nm. Data are the mean ± SD of at least three cultures.

High light

1500

20000

DCW (mg/L)

400

LL-1 dLL-2 dLL-3 dLL-4 d850 nm600

ML-1 dML-2 dML-3 d2800

LL-1 dLL-2 dLL-3 d730 nm

1000

HL-1 dHL-2 dHL-3 dHL-4 d41200

LL-1 dLL-2 dLL-3 dLL-4 d

OD660

6HL-1 dHL-2 dHL-3 dHL-4 dPHA concentration (mg/L)

1400

1015PHA content (wt%)

20Fig. 3. Comparison between PHA contents (wt%) and DCW (mg/L) under low light (8 W/m2) grown cells (left) and high light (50W/m2) grown cells (right). The regression lines and R2 values were shown in each Figure.

Fig. 2. PHA concentrations (mg/L) under different growth light conditions. Cells werecultured under low light (8 W/m2), middle light (20 W/m2: 850 nm) and high light (50 W/m2)
conditions. Data are the mean ± SD of at least three cultures.

◆ PHA production in purple non-sulfur bacteria is related to the cell growth.

PHA production under aerobic conditions➢ Enhancement of PHA production under aerobic condition by addition of acetate• Purple nonsulfur bacteria are known to be able to grow in heterotrophically in the dark under aerobic condition.
We found that R. sulfidophilum, R. imhoffii and R. visakhapatnamense cells hardly accumulated PHA underaerobic condition.
• R. sulfidophilum accumulated 33% of PHA by addition of acetate under aerobic condition and PHA
accumulation was not seen by the addition of reductants, suggesting that acetate-dependent pathway is
involved in PHA accumulation.

➢ Gene expression analysis• Gene expression analysis revealed that the expression of isocitrate dehydrogenase in thetricarboxylic acid (TCA) cycle decreased under aerobic condition and increased by the addition ofacetate, indicating that the TCA cycle activity is involved in PHA production under aerobic condition.
1.82PDH

1.6

1.640

1.2
10.8

Pyruvate

0.60.4

30

0.2

0.8
0.60020

Aerobic Anaerobic

Aerobic Anaerobic Aerobic+ Acetate

Aerobic+ Acetate

AcetylCoA

Acetate0Acetyl-CoAsynthase (ACS)AcetoacetylCoA

3HB-CoA

3HB

PHA synthase(PhaC)

CitrateOxaloacetate

6R. visakhapatnamense

Isocitrate

ACS

Isocitrate dehydrogenase (IDH) 20

Aerobic

IDH

Fig.5. Effect of reductants and carbon sources on PHA production under aerobic
condition. Cells were cultured under aerobic condition in the presence of malateand pyruvate supplemented with DMA, TMA and thiosulfate as reductants andsodium bicarbonate, succinate and acetate as carbon sources. .

Fold change

18

Growth medium: 0.3% sodium malate (Mal)0.3% sodium pyruvate (Pyr)DMS: Dimethylsulfoxide (C2H6S)
TMA: Trimethylamine (C3H9N)
Thiosulfate (S2O32-)
Fig.4. PHA contents under different oxygen growth conditions. Cells werecultured in 25 ml vial under aerobic and oxygen-limited conditions.

10.2Pyruvate dehydrogenase (PDH)

5Oxygen-limited

1.2416Malate

3TCA cycle2-Oxoglutarate

21

Fold change

Aerobic

Oxygenlimitred

Aerobic

Oxygenlimitred

Aerobic

OxygenlimitredR. imhoffii

1.40.4

10R. sulfidophilum

Fold change

Fold change

50

PHA (wt%)

PHA (wt%)

1.4

1816141210864
20

PhaC

1.8

14
12108
640
Aerobic
Aerobic Anaerobic Acetate+ Acetate

Fumarate

Succinyl-CoA

20Succinate

Aerobic
Aerobic Anaerobic Acetate+ Acetate◆ Modification of metabolic pathway lead to PHA accumulation under aerobic condition in marine purple nonsulfur bacteria.
References

1. Higuchi-Takeuchi M, Numata K. Method for the facile transformation of marine purple photosynthetic bacteria using chemically competent cells. MicrobiologyOpen (2019) e953.
2. Higuchi-Takeuchi M, Numata K. Acetate-inducing metabolic states enhance polyhydroxyalkanoate production in marine purple non-sulfur bacteria under aerobic conditions. Front Bioeng Biotechnol. (2019) 28;7:118.
3. Higuchi-Takeuchi M, Numata K. Marine purple photosynthetic bacteria as sustainable microbial production. Front. Bioeng. Biotechnol. (2019). doi.org/10.3389/fbioe.2019.00258