Pathway to scale-up production of duckweed: sustainability, experience and progress.

Eric Lam

Department of Plant Biology & Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, NJ

2nd Int’l Conference on Duckweed Research & Applications SEBS, Rutgers University August 23rd, 2013

Municipal Wastewater

Harvested High-Starch/Protein

Duckweed

Early Effluent

Duckweed Farming

Saccharification Fermentation

Fuel and/or

Feed

Cleaner Water

Duckweed as a Simple Bifunctional Platform for Fuel / Feed Production and Wastewater Treatment

Future vision Future vision

Optimization parameters for duckweed biomass-to-fuel pipeline: integration of biology and engineering.

Biomass production

Post-harvest processing •  Storage •  Drying •  Pressing •  Grinding

Starch solubilization and saccharification •  Autoclaving •  Boiling •  Enzymes •  Time •  Temperature

Fermentation •  Microorganisms •  Density and composition of fermentation mixture •  Throughput time •  Ethanol detection and distillation

Duckweed germplasm development: Collection, characterization, optimization for feedstock production rate as well as target products.

In the field In the lab

Today’s topics

1.  Maintaining and utilizing the world’s largest duckweed collection to identify optimal duckweed strains for different targeted applications.

2.  Learning to farm duckweed on wastewater sites: planting and harvesting.

3.  Preparation for systematic scale-up.

•  Started with 538 isolates/clones from the 5 genera that we received from Biolex, which originated from Landolt’s collection in the ETH, Switzerland; collected from various locations in the world. Together with other strains from others, we started with a total of 584 isolates/clones.

•  Spirodela: 46; Lemna: 279; Landoltia: 46; Wolffia: 122; Wolffiella: 91

•  Currently we have 800+ accessions with additional ones that were recently transferred in 2011 from the collection in Switzerland maintained by Elias Landolt. We have recently added another ~150 characterized strains from the collection of the Chinese Academy of Biology (Chengdu) in 2012.

•  Mission & Vision: to facilitate and broaden the use of Duckweed through sharing and characterization of natural diversity at the genome and phenome levels

Duckweed Futures: germplasm for desirable traits. :

The Rutgers Duckweed Cooperative at the SEBS, Rutgers Est. September 2009 (http://ruduckweed.org/)

What is the impact of duckweed growth on wastewater quality? Comparative analyses of different duckweed strains on various wastewater sources – ammonia, nitrate and phosphate levels.

Duckweed as wastewater remediator.

Wastewater comparison: five different sources

Horticulture waste water Pinelands Nursery, NJ

Municipal wastewater United Water Princeton Meadows, NJ Pine Mountain Lake, Dongguan

Municipal wastewater

Huang Jiang facility stage 2, Dongguan Municipal wastewater

Huang Jiang facility stage 3, Dongguan Municipal wastewater

**Highest in N and P content

PN PM SSH

HJ2 HJ3

Testing the wastewater remediation ability of duckweeds

Wastewater sample: 100 mL Duckweed starting amounts: 1 g. Duration of growth in greenhouse: 2 weeks Compared different strains of duckweed

PN PM SSH HJ2 HJ3

Effects of duckweed growth on PM wastewater.

Ammonia Nitrate Phosphate

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0

4

8

12

16

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L A

mm

onia

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L N

itrat

e

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osph

ate

0 – wastewater without duckweed

Duckweed strain Duckweed strain Duckweed strain

Effects of duckweed growth on HJ#2 wastewater.

Ammonia

0

5

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0 1 2 3 4

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L A

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Nitrate

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L N

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Phosphate

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L Ph

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0 – wastewater without duckweed

Duckweed strain Duckweed strain Duckweed strain

best poor Biomass accumulation:

Different removal efficiency of phosphate by duckweeds.

PN PM

SSH HJ2 HJ3

Huang Jiang #2 Huang Jiang #3

Today’s topics

1.  Maintaining and utilizing the world’s largest duckweed collection to identify optimal duckweed strains for different targeted applications.

2.  Learning to farm duckweed on wastewater sites: planting and harvesting

3.  Preparation for systematic scale-up.

Jan. 20, 2011

April 14, 2011

The Pinelands Nursery Fertilizer Run-off Pond

The Pinelands Nursery site

June 16, 2011

August 30, 2011

Small scale barrier system for on-site testing and harvesting.

Results from 5 sequential harvests at ~75% June 29 to Aug. 22, 2011; once every 2 weeks

Average weight of duckweed collected at about 75% (wet weight): 16.8 + 6 kg Approximate internal surface area of barrier: 160 sq. ft. Estimated total surface area of the pond at Pinelands Nursery: 38,000 sq. ft. or 0.353 hectares

= 3,390 kg (~4 metric tons) of potential duckweed biomass per harvest

Annual expected yield over a 6 month growing season: ~48 metric tons wet weight Total productivity rate: 136 metric tons wet weight/hectare/year or ~6.8 metric tons dry mass/hectare/year With a strain that contains 40% by weight in starch and a fermentation efficiency of 95%, this can potentially produce up to 1.3 tons of ethanol per hectare per year.

First statistics on Pinelands Nursery Site

Successful replacement of Lemna with Spirodela

Spring to Summer 2012 at Pinelands Site

October, 2012

June, 2013

Successful return of planted duckweed to the test site.

Today’s topics

1.  Maintaining and utilizing the world’s largest duckweed collection to identify optimal duckweed strains for different targeted applications.

2.  Learning to farm duckweed on wastewater sites: planting and harvesting

3.  Preparation for systematic scale-up.

Plant lines

Seeding Stocks

Dried Seeds Crop Production

Paths for scale up of terrestrial vs. aquatic crops.

terrestrial crops

aquatic crops

Field Scale (tons/106 grams)

Requirements for robust platform •  Dependable throughput •  Economical •  Flexible scaling •  Reliable purity

mid-range scale (kilogram range)

mini-scale (grams range)

Example: Seeding culture pond for algae biocrude pilot plant of Saffire Energy in New Mexico.

Industrial Algae Cultivation: an aquatic cropping example.

Greenhouse model for a Seeding Stock facility.

30 Liter trays

Total tray surface area: 100 m2 = 0.01 hectares

Total volume of media needed: 12,000 Liters per seeding; assuming changed once per month: 144,000 L. of media needed per year. Based on known potential of duckweed of 100 ton/h/y dw; targeted production capacity: ~1 ton dry mass of duckweed per year.

Defined growth media consideration: cost.

Based on a final N level of 500 ppm

Defined growth media consideration: growth rate.

Estimated cost/yr. HP: $11,600 20-20-20: $971 17-3-17: $1,160

Academic

Social Industrial

The Tripartite Linkage that will Facilitate Development and Acceptance of the Duckweed Platform

"   Financial support by the School of Environmental and Biological Sciences, Rutgers University, NSF-IGERT "   Maintenance and characterization of strains in the Duckweed Cooperative: Ryan Gutierrez, Kenny Acosta, Cyrus Garcia, Nikolai

Borisjuk, Yi Zhang, Jean Wang "   Biofuel and wastewater treatment:

"   Nikolai Borisjuk, Philomena Chu, Jessica Kretch, Deepak Khanna, Ryan Integlia, David Byrnes

"   Duckweed physiology and morphology, sharing of strains and info: "   Dr. Klaus Appenroth - University of Jena, Germany "   Dr. Hai Zhao – CIB, Chengdu, China

"   Duckweed – genomics and genotyping: "   Ryan Gutierrez, Nikolai Borisjuk, Philomena Chu, Hanzhong Zhang "   Beijing Genome Institute (Contract Service) "   Duckweed informatics Todd Michael, Todd Mockler, Douglas Bryant (IBIS Biosci.,

Danforth Plant Science Center) "   Duckweed cultivation, harvesting and processing (Agronomy)

Michelle Low, Tyler Wibbelt

Acknowledgement and Collaborators

Thank you !!!