Cocultivation of Algae and Bacteria for Improved Productivity and Metabolic Versatility

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1 Cocultivation of Algae and Bacteria for Improved Productivity and Metabolic Versatility Pacific Rim Summit on Industrial Biotechnology and Bioenergy October 10-12, 2012 Vancouver, Canada

2 Axenic Cultures in Algal Biotechnology A. Gene/pathway inactivation 3-PGA Current approaches use axenic (pure) cultures of microalgae and/or cyanobacteria Photosynthesis Fatty acids TAGs Carbohydrate (starch, glycogen) storage Productivity is manipulated by imposing environmental or genetic perturbations Examples: B. Photosynthesis Monomer blocks for growth (nucleotides, amino acids, etc) Nutrient limitation -N 3-PGA Storage polymers (carbohydrates, lipids) A) Inactivation of competing pathways to redirect flux towards specific products B) Nutrient (N, S) limitation to inhibit growth and enhance storage product accumulation

3 Axenic Culture Challenges Photosynthesis Lipids CO 2 delivery Storage ACC RuBisCo O 2 removal 3-PGA Hydrocarbons Growth Process engineering: mass-transfer limitations involving gaseous substrate delivery (CO 2 ) and product removal (O 2 ) Growth physiology: balance the energy input with the downstream biosynthetic processes (growth vs. storage compounds) Metabolic engineering: coordination of various pathways needed; changes in expression and/or activity levels may have unanticipated secondary consequences upon product yields. Some functions are subject to product inhibition or allosteric regulation (e.g., RuBisCo photorespiration; acetyl-coa carboxylase regulation by palmitoyl-coa).

4 Co-Existence of Algae & Bacteria in Nature Photoautotroph Heterotroph CO 2 Algae and cyanobacteria use sunlight and CO 2 and produce O 2 and C org molecules that support growth of heterotrophic bacteria Photosynthesis Other anabolism O 2 Carbohydrate polymers org. C Carbohydrates Carbohydrate polymers 3C, 4C intermediates NADH Micronutrients Biomass, other respiration/fermentation products Heterotrophic bacteria provide intrinsic stability and support growth of phototrophs by removing excess O 2, increasing micro-nutrient availability, vitamin biosynthesis Algae-bacterial associations represent metabolically interactive, selfsustaining communities, which display adaptation to a range of harsh conditions

Phototroph-Heterotroph Co-Cultures Metabolic coupling: O 2 produced by the algae is consumed by the heterotroph making stoichiometric amount of CO 2 through oxidation of (endogenous or exogenous)

5 Phototroph-Heterotroph Co-Cultures Metabolic coupling: O 2 produced by the algae is consumed by the heterotroph making stoichiometric amount of CO 2 through oxidation of (endogenous or exogenous) organic C. Stoichiometric constraints drastically increase the intrinsic stability. Heterotrophic bacterium Advantages: -Improved mass transfer & productivity - Increased range of carbon sources - Modularity & ability to spatially separate the processes of light & CO 2 capture with the downstream photosynthate conversion Phototroph (microalga, Cyanobacterium) 5

6 Coupling through Photosynthate Secretion CO 2 Photosynthesis Other anabolism Cellulose O 2 Other carbohydrates? Glucose ADPGluc Glycogen 3C, 4C intermediates NADH Synechococcus sp. PCC 7002 G3P + ADPGluc Gluc6P + UDPGluc Biomass, other respiration/fermentation products Glucosylglycerol Sucrose Sucrose Glucosylglycerol Rationale: Redirect fixed CO 2 to mono/ disaccharide derivatives, which can be excreted and used as a carbon and energy source for biofuel synthesis by hetrotrophic organisms. Approach: Eliminate glycogen storage by mutation of glga1, glga2, and glgb, and/or glgc but maintain high photosynthetic rate. 6 In collaboration with Bryant s Lab (Penn State)

Glucose, sucrose and glucosylglycerol are excreted in glg

7 Engineering Glycogen Metabolism to Increase Carbohydrate Excretion This strategy works! Glucose, sucrose and glucosylglycerol are excreted in glg mutants of Synechococcus sp. PCC 7002 that cannot make glycogen. 7 Bryant, Xu et al., 2012 (in prep)

8 Metabolic Coupling through Secreted C - Heterotrophic growth supported through secretion of sugars and osmolytes (>300hs) - Biomass concentration can be manipulated by varying growth conditions (light, CO 2 ) - Plug-and-play approach in which process of photosynthetic carbon fixation and product biosynthesis is spatially separated Module A: CO 2 -> C org (sugars, organic acids) Module B: C org -> target bio-product 8

Metabolic Coupling via Exogenous C Organic carbon (waste) Heterotrophic bacterium O 2 CO 2 Phototrophic algae or cyanobacteria Solar energy Biomass, value-added products - Allows utilization of

productivity and growth rates Co-culture (10 mm lactate, 5mM HCO 3-, 50 rpm) Heterotroph (10 mm lactate, 5mM HCO 3-, 50 rpm) Phototroph (5mM HCO 3-, 250 rpm) Phototroph (5mM HCO 3-, 50 rpm) -

9 Metabolic Coupling via Exogenous C Organic carbon (waste) Heterotrophic bacterium O 2 CO 2 Phototrophic algae or cyanobacteria Solar energy Biomass, value-added products - Allows utilization of various C sources (including waste streams) - Limited mass transfer as O 2 and CO 2 are produced throughout cultivation vessel - Axenic (pure) cultures display significantly lower biomass productivity and growth rates Co-culture (10 mm lactate, 5mM HCO 3-, 50 rpm) Heterotroph (10 mm lactate, 5mM HCO 3-, 50 rpm) Phototroph (5mM HCO 3-, 250 rpm) Phototroph (5mM HCO 3-, 50 rpm) - Co-culture displays higher growth & productivity; does not need high mass transfer rates ; utilizes both carbon sources; no O 2 accumulation - Ratio of C org /CO 2 affects the proportion heterotroph & phototroph biomass

10 Waste Treatment using Algal Co-cultures

11 Wastewater Treatment: Setup Wastewater with high concentration of complex carbohydrates, N, and P Co-culture Bacilllus sp. and Haematococcus pluvialis Light, no bubbling, low agitation

12 Wastewater Treatment: COD Results after 200 hr incubation: Untreated wastewater 100% Treated wastewater 46% 29%

13 Wastewater Treatment: Nitrogen Results after 200 hr incubation: Untreated wastewater Treated wastewater (10 days)

14 Production of High-Value Biomass Results after 200 hr incubation: Biomass: 2.2 g/l Algae: 1.4 g/l Astaxanthin: ~ 0.8% START END

15 Astaxanthin Accumulation Value proposition: - waste treatment (reduction in COD/BOD, N, P) - high-value biomass production - reduced masstransfer, energy expenditures, as well as C emissions

16 Summary Phototroph-heterotroph co-cultures present an alternative option for photosynthetic production of value-added products and commodities such as biofuels. In comparison to axenic (pure) cultures, co-cultures display broader substrate versatility, higher productivities due to decreased of mass transfer requirements, and provide increased engineering flexibility by spatially and/or temporally separating the processes of photosynthesis and photosynthate conversion We have successfully applied co-cultivation of heterotrophic bacteria with microalgae for wastewater treatment and production of high-value biomass. The approach opens new ways for designing highly-efficient production processes for feedstock biomass production as well as allows utilization of variety of organic agricultural, chemical, or municipal wastes. 16

17 Acknowledgements Pacific Northwest National Lab: Dr. Gregory Pinchuk Eric Hill Leo Kucek Dr. Sergey Stolyar Dr. Oleg Heidebrecht University of Wisconsin: Trang Vu Dr. Jennifer Reed Penn State University: Dr. Donald Bryant Dr. Gaozhong Shen Dr. Yu Xu Funding by: U.S. DOE BER through Genomic Sciences Program PNNL LDRD and Technology Maturation programs Burnham Inst. Medical Research: Dr. Andrei Osterman Dr. Jessica DeIngenis 17

Photosynthetic production of biofuels from CO 2 by cyanobacteria using Algenol s Direct to Ethanol process Strain development aspects

Photosynthetic production of biofuels from CO 2 by cyanobacteria using Algenol s Direct to Ethanol process Strain development aspects Paul Roessler - Oct 1, 2014 Algal Biomass Summit Algenol Overview Advanced