Bioenergy from Photosynthetic Microorganisms: What are the Basic Research Needs? ���

National Science Foundation ���Nov. 21, 2011���

���Peter Lammers ���

New Mexico State University���

Input Logistics for Photosynthetic Microorganisms

Light, carbon dioxide, nitrogen and phosphorus – Major gaps in knowledge about N and P uptake,

assimilation, storage and release – Nutrient limitation triggers major metabolic shifts

leading to lipid and/or polysaccharide accumulation – C, N, P competition are primary drivers of aquatic

ecology –  Extreme taxonomic diversity across major groups of

photosynthetic organisms under consideration

Waste as Renewable Resource •  N and P dependent eutrophication: a legacy of

the green revolution •  dead zones in oceans and groundwater

leaching from dairy/feedlot lagoons, landfills, receiving little or no treatment

•  Estimated 2 billion humans with inadequate access to waste treatment/sanitation

•  Animal wastes contain C, N, P in ratios not far from the Redfield ratio

Animal Waste The phosphorus lost to surface and ground water from animal waste is equivalent to ~ 40% of mined phosphorus. Rittmann, B.E., Mayer, B., Westerhoff, P., Edwards, M. 2011. Capturing the lost phosphorus. Chemosphere, 84(6), 846-853.

Population pressures, public health concerns, energy efficiency and resource recovery needs will be major design drivers of next generation systems. Mara, D. 2001. Appropriate Wastewater Collection, Treatment and Reuse in Developing Countries. Municipal Eng, 145(4), 299-303.

Must identification of chemical inhibitors and their metabolic targets in animal waste streams. Kolpin, et al. 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999-2000: A national reconnaissance. Environmental Science & Technology, 36(6), 1202-1211. Richardson, S.D. 2009. Water Analysis: Emerging Contaminants and Current Issues. Analytical Chemistry, 81(12), 4645-4677.

Cultivation methods for > 5 gdw/L biomass at > 50% lipid content with low nutrient consumption in a small-scale closed system demonstrated.

Max cell density achieved outdoors in PBR = 6 gdw/L

Min outdoor inoculation density tested in outdoor PBR = 0.25 gdw/L

Maximum cell density achieved indoor PBR @ 1800 µE > 15 gdw/L

Nannochloropsis cultivation metrics

y = 0.2294x + 0.0812 R² = 0.9833

0

1

2

3

4

5

0 5 10 15 20

gdw

/L

Days Cultivation

N. Salina (CCMP 1776) Linear Growth Rate 6X nutrients (N=3)

Ave g/L

Linear (Ave g/L)

706 Batch FAME tests •  Mean % Lipid = 34.7 •  Std Dev = 10.8 •  Min = 9.6% •  Max = 65.4%

Loca%on Species Days  in  opera%on

#  Batch  Transfers

Start  Date End  Date

Fort  Collins N.  oculata 176  days 25 4/3/08 9/26/08 Fort Collins N.  oculata 288  days 22 8/6/08 5/21/09 Fort Collins N.  oculata 131  days 16 5/6/09 9/14/09 Fort Collins N.  salina 161  days 29 10/30/09 4/9/10 Fort Collins N.  salina 122  days 41 3/17/10 7/7/10 Fort Collins N.  salina 144  days 11 6/10/10 11/1/10 Coyote  Gulch

N.  oculata 260  days 10 7/16/09 4/2/10

Coyote Gulch

N.  salina 421  days   20   10/5/09 N/A

Nannochloropsis strains are very stable in serial batch cultures over periods of years in Solix PBR systems

Shotgun Lipidomics of Nannochloropsis salina by Ultrahigh Resolution FT-ICR Mass Spectrometry Reveal Serious Gaps in

Fundamental Biochemistry

Omar Holguin Tanner Schaub Wayne VanVoorhies Peter Lammers

m/z1,2001,1001,000900800700600500400

Nannchloropsis Lipid Extract, FT-ICR Mass Spectrum Negative Ion Mode

1375 compounds identified

New Mexico State University

Long Chain Sulfate Lipid Species Detected by FT-ICR-MS

OTU ID Taxonomy OTU

ID Taxonomy 5 Stappia 370 Roseovarius 7 Geminicoccus 372 Psychroserpens 38 Maribacter 391 Fangia 39 Phyllobacteriaceae 417 Coenonia 49 Alphaproteobacteria 445 Verrucomicrobia 56 Psychroserpens 465 Gilvibacter 63 Propionibacterium 469 Rhodobacteraceae 65 Fangia 488 Chrysiogenes 81 Bacteriovoracaceae 490 Aurantimonas 82 Bacteroidetes 498 Gilvibacter 112 Gilvibacter 508 Chrysiogenes 140 Phyllobacteriaceae 510 Cryptomonadaceae 150 Aeriscardovia 511 Thalassobaculum 162 Bacteria 515 Bacteria 170 Parvibaculum 517 Campylobacterales 176 Chrysiogenes 519 Filifactor 178 Cryptomonadaceae 526 Proteobacteria 222 Corynebacterium 538 Psychroserpens 246 Filifactor 541 Phyllobacteriaceae 267 Psychroserpens 558 Flammeovirgaceae 270 Gilvibacter 568 Roseovarius 279 Alphaproteobacteria 587 Kordiimonas 280 Coenonia 591 Phyllobacteriaceae 299 Coenonia 604 Campylobacterales 323 Roseovarius 623 Chrysiogenes 324 Chrysiogenes 634 Larkinella 341 Mycoplasmataceae 640 Maribacter 354 Cryptomonadaceae 676 Gilvibacter 360 Chrysiogenes 695 Bacteria 362 Fangia 696 Geminicoccus 363 Chrysiogenes 704 Stappia

The core algal-symbiome during scale up reveals dramatic, dynamic diversity in heterotrophic component of Nannochloropsis in industrial cultures. 16S metagenomic project – NSF travel grant to Scott Fulbright for collaboration with J. Xu team at QIBEBT, Qingdao, China 2010

Nannochloropsis ���

•  Needs –  Metabolic flux

analysis –  C, N, P, uptake,

assimilation and storage/release

Small Genomes – 24-29 Mbp Seven genomes in queue for public release J. Xu QIBEBT ~3500 common “pan-genome” ~35,000 genes not shared across the seven genomes

Transformation methods in progress Strong public track record for cultivation success High natural oil content