Theme III: Cost-effective conversion of biomass into biofuels and co-products.

Transcription

1 Ecology and Physiology of Algal Biodiesel Production Dr. Mark A. Schneegurt, Wichita State University Theme III: Cost-effective conversion of biomass into biofuels and co-products.

2 Proposed Work on Open Cultures Identify and obtain suitable existing algal cultures Develop lab-scale and pilot-scale algal culture systems Monitor oil and biomass production, with changing conditions Define microbial community structure using DNA fingerprinting (DGGE) and sequencing Is the trajectory of community assembly consistent? Are there windows where assembly is best manipulated? What is the source of competing algae and others? What environmental conditions foster or inhibit community shifts? Can communities be managed through process and startup schemes?

3 Objectives and Progress Identify and obtain suitable existing algal cultures Isolates chosen from NREL Aquatic Species Program collection Nanochloropsis (green alga), Nitzschia (diatom) Cultures obtained from University of Hawaii Maintenance conditions established in laboratory Develop lab-scale up to pilot-scale algal culture systems Shake-flask cultures examined for minimal media requirements Bioreactor cultures (4 L) established Open trough cultures (1.5 L) maintained in greenhouse Pilot-scale (100 L) outdoor open ponds established as batch cultures Monitor oil and biomass production, with changing conditions Culture density followed with cell counts and pigment analysis Bulk biomass samples retained for oil analysis Define microbial community structure using DNA fingerprinting (DGGE) and sequencing DGGE conditions established for three rrna primer sets Universal bacterial, universal eukaryotic, phototroph-specific Metagenomic DNA directly extracted from open-pond cultures rrna PCR amplified and subjected to DGGE fingerprinting Amplicon patterns comparable using numerical taxonomy techniques Bands excised, re-amplified, and sequenced for identification

4 NREL Aquatic Species Program US Department of Energy's Office of Fuels Development conducted a long-term study on the use of algae for the production of transportation fuels ending in A microscopic view of Nannochloropsis spp. Generated over 3000 characterized algal strains with high lipid production.

5 Objectives and Progress Identify and obtain suitable existing algal cultures Isolates chosen from NREL Aquatic Species Program collection Nanochloropsis (green alga), Nitzschia (diatom) Cultures obtained from University of Hawaii Maintenance conditions established in laboratory Develop lab-scale up to pilot-scale algal culture systems Shake-flask cultures examined for minimal media requirements Bioreactor cultures (4 L) established Open trough cultures (1.5 L) maintained in greenhouse Pilot-scale (100 L) outdoor open ponds established as batch cultures Monitor oil and biomass production, with changing conditions Culture density followed with cell counts and pigment analysis Bulk biomass samples retained for oil analysis Define microbial community structure using DNA fingerprinting (DGGE) and sequencing DGGE conditions established for three rrna primer sets Universal bacterial, universal eukaryotic, phototroph-specific Metagenomic DNA directly extracted from open-pond cultures rrna PCR amplified and subjected to DGGE fingerprinting Amplicon patterns comparable using numerical taxonomy techniques Bands excised, re-amplified, and sequenced for identification

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7 Simplifying the Recommended Medium for Larger Cultures Media composition Growth Media Composition Growth Without NaCl Yes Without NaHCO 3 Yes Without NaH 2 PO 4 Yes Without NaNO 3 No Without Fe/EDTA Yes Without Trace Metal Yes Without MgCl 2.6H 2 O Yes Without HEPES Yes Without MgSO 4.7H 2 O Yes Without Vitamins Yes Without NaSiO 3.9H2O Yes Without CaCl 2 No Without KCl No Without H 3 BO 3 Yes With Tap Water +CaCl 2 + NaNO 3 +HEPES+ KCl Yes With Tap Water + rest of the media Yes With Miracle Grow NaCl+ CaCl 2 Yes Without Trace Metal + Vitamins + HEPES + Fe/EDTA Yes

8 Objectives and Progress Identify and obtain suitable existing algal cultures Isolates chosen from NREL Aquatic Species Program collection Nanochloropsis (green alga), Nitzschia (diatom) Cultures obtained from University of Hawaii Maintenance conditions established in laboratory Develop lab-scale up to pilot-scale algal culture systems Shake-flask cultures examined for minimal media requirements Bioreactor cultures (4 L) established Open trough cultures (1.5 L) maintained in greenhouse Pilot-scale (100 L) outdoor open ponds established as batch cultures Monitor oil and biomass production, with changing conditions Culture density followed with cell counts and pigment analysis Bulk biomass samples retained for oil analysis Define microbial community structure using DNA fingerprinting (DGGE) and sequencing DGGE conditions established for three rrna primer sets Universal bacterial, universal eukaryotic, phototroph-specific Metagenomic DNA directly extracted from open-pond cultures rrna PCR amplified and subjected to DGGE fingerprinting Amplicon patterns comparable using numerical taxonomy techniques Bands excised, re-amplified, and sequenced for identification

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11 Objectives and Progress Identify and obtain suitable existing algal cultures Isolates chosen from NREL Aquatic Species Program collection Nanochloropsis (green alga), Nitzschia (diatom) Cultures obtained from University of Hawaii Maintenance conditions established in laboratory Develop lab-scale up to pilot-scale algal culture systems Shake-flask cultures examined for minimal media requirements Bioreactor cultures (4 L) established Open trough cultures (1.5 L) maintained in greenhouse Pilot-scale (100 L) outdoor open ponds established as batch cultures Monitor oil and biomass production, with changing conditions Culture density followed with cell counts and pigment analysis Bulk biomass samples retained for oil analysis Define microbial community structure using DNA fingerprinting (DGGE) and sequencing DGGE conditions established for three rrna primer sets Universal bacterial, universal eukaryotic, phototroph-specific Metagenomic DNA directly extracted from open-pond cultures rrna PCR amplified and subjected to DGGE fingerprinting Amplicon patterns comparable using numerical taxonomy techniques Bands excised, re-amplified, and sequenced for identification

12 DGGE Gel of PCR Amplicons from Open-pond Metagenomic DNA Extracts Black = bacterial primers Red = eukaryotic primers Samples were from 100 L ponds taken at different points in the batch cycles.

13 DGGE Gel of PCR Amplification of Open-pond Samples and Excised Products Red = eukaryotic primers Black = phototroph primers Metagenomic DNA from samples of 100-L openponds. After PCR amplification and DGGE separation, bands were excised, re-amplified, and run again on DGGE. These bands are being sequenced to confirm identity.

14 DGGE Gel of Amplicons from Open-pond Samples and KU Reactors Black = eukaryotic primers Red = phototroph primers Samples from 100-L openponds are at right. Samples from KU wastewater plant open reactors are at left.

15 Prospects Better understanding of community assembly in open algal ponds Application of molecular ecology techniques for pond monitoring Developing baseline pond systems for study of harvesting technology Attract industry and exploit rangeland resources in Kansas Develop sustainable and inexpensive local farm-scale systems Involve graduate and undergraduate students in bioenergy research Expose pre-college students and teachers to bioenergy research