Enhanced photosynthesis, biomass production and nutrient uptake

of duckweeds by plant growth-promoting bacterium,

Sinorhizobium sp. SP4

The Second International Conference on Duckweed Research and Applications August 23 2013

Tadashi TOYAMA, Yasuhiro TANAKA, Kazuhiro MORI (University of Yamanashi)

Masaaki MORIKAWA (Hokkaido University)

Enhanced photosynthesis, biomass production and nutrient uptake of duckweeds

by plant growth-promoting bacterium (PGPB), Sinorhizobium sp. SP4

This study is an answer to the questions

(1) “ Can PGPB enhance duckweed activity?”

(2) “Can PGPB improve nutrient removal and starch-biomass production from wastewater by duckweeds?”

Outline

1.  Introduction: Background information and our objectives

2. Answer to the first question. “Can PGPB enhance

duckweed activity?” 3. Answer to the second question. “Can PGPB improve

nutrient removal and starch-biomass production from wastewater by duckweeds?”

4. Conclusion:

Our idea for application of duckweed-PGPB symbiotic relationship to wastewater treatment and production of energy/chemical feedstock.

Duckweed aquaculture system

• Duckweed biomass has high starch content (~70%) and low cellulose, hemicellulose and lignin contents (~10%). • Duckweed biomass is also a good source for proteins.

 Role of duckweed (1): Good alternative starch feedstock for bio-ethanol

• Duckweed can take up nutrients (N and P) and heavy metals, and duckweed and its rhizosphere bacteria can degrade toxic organic chemicals.

 Role of duckweed (2): Water purification and wastewater treatment

 Dual role of duckweed: Nutrient removal from wastewater and at the same time production bioenergy • The co-benefit system combining nutrient removal and starch-biomass production from wastewater by using duckweed will be a key technology for a low-carbon and sustainable recycling society. • However, the major bottleneck is the slowness of plant growth and reactions.

CO2H2O CO2+

O2

Photosynthesis

N

N P

CHOCHO

O2

P Take up nutrient for growth“Production of plant biomass”

Duckweed aquaculture system

NC State University

• Wastewater, • Sewage effluent, • Agricultural effluent…

Starch-biomass Protein-biomass

Purified water

Bio-energy/chemical feedstock Feeds

Bio-ethanol, bio-gas, proteins

Plant Growth-Promoting Bacteria (PGPB) • Some PGPB colonize rhizosphere or endosphere of plants. • PGPB can stimulate plant growth, increase yield and reduce biotic or abiotic plant stress.

Mechanisms: (1) Production of phytohormones (2)N2 fixation (3) Solubilization of nutrients (P) and/or minerals (Fe) (4) Antagonism against phytopathogenic microbes

Online. APSnet Features. doi: 10.1094/APSnetFeature/2005-0605

Biological Control 59 (2011) 114–122

Control pepper plant PGPB-pepper plant Control rice PGPB-rice

Objectives of this study

We adopted plant growth-promoting bacteria (PGPB) as breakthrough strategy for the bottleneck of duckweed system.

We tried to overcome the slowness of plant growth and reactions by PGPB.

The strategy is an alternative technique to genetic modification.

1. To examine the effects of PGPB on photosynthesis, biomass growth, nutrient uptake and starch-biomass production of duckweed.

2. To demonstrate the accelerated nutrient removal and starch-biomass production from wastewater by duckweed.

Flow chart of this study

1 cm

In this study, we adopted giant duckweed (Spirodela polyrhiza) as model duckweed.

1.  We isolated PGPB candidate bacterial strains from Spirodela roots.

2.  We screened and selected the most effective PGPB. 3.  We examined effects of PGPB on Spirodela activities

(growth, photosynthesis and plant metabolome). 4.  We demonstrated the accelerated nutrient removal and

starch biomass production from wastewater by using Spirodela and PGPB.

5.  We examined effects of PGPB on other duckweeds (Lemna minor and Wolffia arrhiza)

Answer to the first question.

“Can PGPB enhance duckweed activity?”

Isolation of PGPB and PGP assay

Co-culture of sterilized Spirodela with environmental waters

Isolation of bacteria from the root surfaces

● ● ●

● ● ● ●

Plant growth-promoting (PGP) assay

• Environmental water: River waters and effluent of sewage treatment plant • During this co-culture, native bacteria in environmental water colonized the Spirodela roots

• Roots were washed 3 times • Bacteria attaching roots were collected by sonication and vortex • Plating on 1/100 TS agar

• Sterile Spirodela (5 plants) and each isolate (OD600=0.1) were co-cultured in sterile Hoagland solution for 7 days. • Bacteria-free sterile Spirodela was used for control test. • Biomass growth (dry weight) was measured.

Isolation of PGPB and PGP assay

Dr. Morikawa

PGP effects of isolated bacterial strains

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Sterile-Spirodela control

Some of isolated bacterial strains

• Isolated bacterial strains from Spirodela roots showed various PGP effects on Spirodela.

Sinorhizobium sp. SP4 • Sinorhizobium sp. SP4 showed the highest plant growth-promoting effect on Spirodela.

• SP4 was isolated from the roots of Spirodela grown in river water.

Co-culture of Spirodela with strain SP4 for 5 days

Monitor Spirodela activities

• 300 mL Hoagland • 28℃ • 16h-light / 8 h-dark • Photo flux density, 80 µmol/m2/s • Initial Spirodela number, 10 plants • SP4 cell density, 107 cells/mL

1. Growth (plant number) 2. Biomass production (dry weight) 3. Starch content (%) 4. Chlorophylls a and b contents 5. Carbon (C% & δ13C ) and nitrogen

(N% & δ15N) contents 6. Photosynthesis activity 7. Spirodela metabolome

Detailed examination

Effect of SP4 on Spirodela size

• SP4 slightly enlarged Spirodela frond size. • SP4 significantly shorten Spirodela root length. • SP4 slightly reduced the weight of Spirodela.

Sterile Spirodela SP4-Spirodela • Frond (leaf): 7×7 mm • Root: about 30 mm • Dry weight per plant: 0.95 mg

• Frond (leaf): 8×8 mm • Root: about 18 mm • Dry weight per plant: 0.81 mg

10 mm

Effects of SP4 on growth and Chlorophylls content

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Spirodela growth

Sterile Spirodela

Sterile Spirodela

Sterile Spirodela

SP4-Spirodela

SP4-Spirodela

SP4-Spirodela

• SP4 significantly increased growth rate (division or budding rate) after 3 days co-culture. • SP4 significantly and immediately increased chlorophylls content after 1 day co-culture.

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Effects of SP4 on biomass and starch production D

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• Biomass production within 5 days of SP4-Spirodela (114 mg) was 2.3 times more than that of sterile Spirodela (49 mg). • Starch production of SP4-Spirodela (multiplying biomass by starch content) was 2.3 times more than that of sterile Spirodela.

Biomass production Starch content

Biomass Production 49 mg/ 5days

Biomass production 114 mg/5 day

Initial biomass

Biomass on 5th day

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Effects of SP4 on C (13C) and N (15N) contents

% ‰

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Carbon content (%), δ13C (‰) Nitrogen content (%), δ15N(‰)

• C content and δ13C of SP-Spirodela were the same levels as those of sterile Spirodela. • N content and δ15N of SP4-Spirodela were significantly higher than those of sterile Spirodela. The result indicates Spirodela took up organic-N released from SP4.

C% C%

N%

N%

δ13C δ13C δ15N

δ15N

δ15N=(15N/14N of sample / 15N/14N of atmosphere – 1)x1000

Effect of SP4 on photosynthetic activity

・5 days culture SP4-Spirodela and sterile Spirodela were used. ・Temperature: 28℃ ・Photon flux density:

150 µmol/m2/s ・CO2 concentration: 400 µmol CO2/mol

Experimental conditions

Photosynthetic activity per plant

(n mol CO2 / plant / s)

Photosynthetic activity per dry weight

(n mol CO2 / g / s) Sterile Spirodela 0.336 0.480 SP4-Spirodela 0.632 0.903

Photosynthetic system LI-6400XT

• SP4 highly activated photosynthesis of Spirodela. • SP4-Spirodela photosynthetic activity was 1.9 times higher than that of sterile Spirodela.

Spirodela metabolome analysis

• Whole Spirodela sample (fronds and roots) were used for metabolomic analysis.

• The metabolome was extracted by methanol.

• The extracted metabolome was analyzed by CE-TOFMS (capillary electrophoresis：CE)-（time-of-flight mass spectrometry：TOFMS） CE-TOFMS

• 214 metabolic peaks were detected. • Major 108 metabolic peaks were quantified.

Effect of SP4 on Spirodela metabolome

C-0

aC

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0day, 1std, 3rdd, 5thd Sterile Spirodela

1std, 3rdd, 5thd SP4 Spirodela

• SP4 changed Spirodela metabolome immediately and dramatically.

HeatMap of Hierarchical Cluster Analysis (HCA)

Effect of SP4 on chlorophyll synthesis pathway

Time (day) Rat

io o

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SP4-Spirodela / sterile Spirodela > 1: ■ →Increase metabolite levels by SP4

SP4-Spirodela / sterile Spirodela < 1: ■

→Decrease metabolite levels by SP4

0123

1 3 5

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Ala

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Chl. a

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Chl. b

• SP4 activated chlorophyll synthesis pathway.

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DHAP

Effects of SP4 on Calvin cycle and C fixation CO2

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Ru5P

G3P

TCA cycle…

Starch content at 5 d • SP4-Spirodela = 18.5% • Sterile Spirodela = 18.1%

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3-PGA

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F6P

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G1P

• SP4 activated Calvin cycle and C fixation. • Starch content of SP4-Spirodela was almost the same level as sterile Spirodela.

■：Increase by SP4

■：Decrease by SP4

Effects of SP4 on glycolysis pathway and TCA cycle

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G3P

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Pyrvate

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• SP4 increased glycolysis pathway metabolism levels and decreased some TCA cycle metabolism levels.

■：Increase by SP4

■：Decrease by SP4

TCA cycle

Effect of SP4 on N uptake and N metabolism

TAC cycle

NO3– NO2

– NH3 ATP ADP + Pi

Transamidation

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Chlorophylls a and b

• N content of SP4-Spirodela (5.4%) was 2.1 times higher than that of sterile Spirodela (2.6%). • SP4 highly activated N uptake pathway and N metabolism in Spirodela.

■：Increase by SP4

■：Decrease by SP4

This part is answer to the first question.

“Can PGPB enhance duckweed activity?”

Answers

1.  PGPB, Sinorhizobium sp. SP4, was isolated from Spirodela root.

2.  SP4 can strongly enhance growth, biomass production and photosynthesis of Spirodela.

3.  SP4 can dramatically change Spirodela metabolome. 4.  SP4 can change Spirodela into “super activated

Spirodela” .

Answer to the second question.

Can PGPB improve nutrient removal and starch-biomass production from wastewater by duckweeds?

Sequencing batch reactor (SBR) with SP4-Spirodela

• SBR treated 100 mL Hoagland solution (5 mg NO3-N/L, 1 mg PO4-P). • Temperature and light conditions: 28℃,16h-light / 8 h-dark, 80 µmol/m2/s. • Initial Spirodela number: 10 plants in each cycle. • Reaction time: 24 h per cycle.

10 plants were transferred to

fresh flask [1st cycle] [2nd cycle]

10 plants were transferred to

fresh flask [3rd cycle]

Co-culture of Spirodela with strain SP4 for 3 days Spirodela plants were highly activated by SP4 during this co-culture.

Sequencing batch reactor (SBR) for nutrient removal from Hoagland

Activated Spirodela

NO3-N and PO4-P removal and plant growth in SBR

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• NO3-N and PO4-P were removed rapidly in SP4-Spirodela reactor. • Plant growth was promoted in SP4-Spirodela reactor.

Sterile Spirodela reactor

SP4-Spirodela reactor

NO3-N

PO4-P

Plant growth

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PO4-P

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Time (day)

NO3-N and PO4-P removal and plant growth in SBR Nutrient removal rate and plant growth rate

Cycle number

Sterile Spirodela reactor

SP4-Spirodela reactor

Effects of SP4 =ratio of SP4 /

sterile Spirodela

NO3-N removal rate (mg-N/day)

1st 1.26 2.27 1.80 2nd 1.30 2.44 1.87 3rd 1.33 2.51 1.89

PO4-P removal rate (mg-P/day)

1st 0.223 0.848 3.80 2nd 0.226 0.786 3.48 3rd 0.223 0.834 3.73

Increase of plant number (plant/day)

1st 7.83 12.5 1.60 2nd 8.17 12.2 1.50 3rd 7.67 13.7 1.78

• NO3-N and PO4-P removal and plant growth rates in SP4-Spirodela reactor were repeatedly accelerated compared to sterile Spirodela reactor.

Nutrient removal and starch-biomass production from wastewater by SP4-Spirodela

Bacteria

100 plants,

1000 mL of 2nd effluent of sewage treatment plant,

>20˚C, natural sunlight

Non-inoculated Spirodela

SP4-Spirodela (Activated Spirodela)

Nutrient removal and starch-biomass production from wastewater by SP4-Spirodela

Non-inoculated Spirodela

reactor

SP4-Spirodela reactor

Non-inoculated Spirodela

reactor

SP4-Spirodela reactor

Starting date After 2 days

• Plant growth was significantly promoted in SP4-Spirodela reactor.

*Another experiment: 300 plants in 3000 mL effluent of swage treatment plant

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SP4-Spirodela reactor

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NH4-N

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Time (day)

Nutrient removal and starch-biomass production from wastewater by SP4-Spirodela

• NO3-N and PO4-P were removed rapidly in SP4-Spirodela reactor.

Performance in one reactor

Non-inoculated Spirodela

SP4-Spirodela

Effects of SP4 =ratio of SP4/

control NH4-N removal rate (mg/d) 1.87

2.01 1.07

NO3-N removal rate (mg/d) 0.820

1.63 2.00

PO4-P removal rate(mg/d) 0.223 0.535 2.40 Biomass production rate (mg/d)

21.4 51.0 2.38

Starch content in plant (%) 16.6 17.4 1.04 Starch production rate (biomass×starch) (mg/d)

3.55 8.87 2.50

Nutrient removal and starch-biomass production from wastewater by SP4-Spirodela

• Rates of NO3-N and PO4-P removal and starch production from wastewater were more than 2 times higher than control.

This part is answer to the second question.

“Can PGPB improve nutrient removal and starch-biomass production from wastewater by duckweeds?”

Answers

1.  Sinorhizobium sp. SP4 can enhance NO3-N and PO4-P removal from wastewater and at the same time promote production of starch-biomass from wastewater by Spirodela.

2.  However, in this study, SP4 could not enhance NH4-N removal from wastewater by Spirodela

Answer to the additional question.

“Can SP4 enhance other duckweeds (Lemna minor and Wolffia arrhiza) activities?”

PGP effect of SP4 on Lemna minor

• SP4 enlarged frond size and substantially shorten root length.

Sterile Lemna SP4-Lemna

10 mm

Photosynthetic activity per plant (n mol CO2/plant/s)

Photosynthetic activity per dry weight (n mol CO2/g/s)

Sterile Lemna 0.0690 0.345 SP4-Lemna 0.142 (2.1 times higher) 0.711

• SP4 significantly enhanced biomass production, increased chlorophylls contents and enhanced photosynthesis activity of Lemna minor.

PGP effect of SP4 on Lemna minor Biomass production rate (dry weigh mg/ day)

Sterile Lemna 4.76 SP4-Lemna 10.7 (2.3 times higher)

Chlorophyll a content (µg/mg)

Chlorophyll b content (µg/mg)

Sterile Lemna 4.06 1.27 SP4-Lemna 11.5 (2.8 time higher) 3.53 (2.8 times higher)

PGP effect of SP4 on Wolffia arrhiza

• SP4 green Wolffia color. • After 10 days half of sterile Wolffia became turions and sank down to the bottom. However, SP4-Wolffia stayed the vegetative frond at water surface.

Sterile Wolffia SP4-Wolffia

SP4-Wolffia after 10 days

Sterile Wolffia after 10 days

5 mm

Effect of SP4 on Wolffia arrhiza

Photosynthetic activity per plant (n mol CO2/plant/s)

Photosynthetic activity per dry weight (n mol CO2/g/s)

Sterile Wolffia 0.00503 0.252 SP4-Wolffia 0.00954 (1.9 times higher) 0.477

Biomass production rate (dry weigh mg/ day) Sterile Wolffia 5.24 SP4-Wolffia 10.2 (2.0 times higher)

Chlorophyll a content (µg/mg)

Chlorophyll b content (µg/mg)

Sterile Wolffia 8.61 2.35 SP4-Wolffia 14.6 (1.7 time higher) 4.32 (1.8 times higher)

• SP4 significantly enhanced biomass production, increased chlorophylls contents and enhanced photosynthesis activity of Wolffia arrhiza.

Answers

1.  SP4 can enhance plant growth and photosynthesis of other duckweeds (Leman minor and Wolffia arrhiza).

2.  The plant growth-promoting effect of SP4 seems to be highly-versatile effect on duckweeds.

Answer to the additional question.

“Can SP4 enhance other duckweeds (Lemna minor and Wolffia arrhiza) activities?”

Conclusion: Our idea for applications of duckweed-PGPB symbiotic relationship to wastewater

treatment and bio-production

• Wastewater, • Sewage effluent, • Agricultural effluent…

Nutrients in wastewater

Uptake and Photosynthesis

by duckweed

CO2

O2

Starch-biomass

Production of highly activated duckweeds by

PGPB

Supplying activated duckweed

plants

Wastewater treatment tank

Activation tank

Activated duckweed can recover nutrients from wastewater and at the same time produce starch-biomass at high speed.

• Duckweed-PGPB symbiotic relationship will improve the co-benefit system combining nutrient removal and starch-biomass production from wastewater.

• There must be other duckweed-bacteria symbiotic relationships, including other PGPB or other bacteria.

• The duckweed-bacteria symbiotic relationships will open the new door for duckweed systems and strongly support a low-carbon and sustainable recycling society.

Thank you very much.

Conclusion