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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
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• Current approaches use axenic (pure) cultures of microalgae and/or cyanobacteria • Productivity is manipulated by imposing environmental or genetic perturbations • Examples:
A) Inactivation of competing pathways to redirect flux towards specific products B) Nutrient (N, S) limitation to inhibit growth and enhance storage product accumulation
Storage polymers (carbohydrates,
lipids)
Photosynthesis
3-PGA
Monomer blocks for growth (nucleotides,
amino acids, etc)
Nutrient limitation B.
Carbohydrate (starch, glycogen)
storage
Photosynthesis
3-PGA
Fatty acids
Gene/pathway inactivation
TAGs
A.
Axenic Cultures in Algal Biotechnology
-N
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Storage
Photosynthesis 3-PGA
Growth
• Process engineering: mass-transfer limitations involving gaseous substrate delivery (CO2) and product removal (O2) • 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).
Axenic Culture Challenges
CO2 delivery
O2 removal
Lipids Hydrocarbons
RuBisCo
ACC
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Co-Existence of Algae & Bacteria in Nature
Carbohydrate polymers
CO2
Photosynthesis
Carbohydrates
Carbohydrate polymers
O2
Other anabolism
3C, 4C intermediates
NADH
Biomass, other respiration/fermentation
products
org. C Micro-
nutrients
• Algae and cyanobacteria use sunlight and CO2 and produce O2 and Corg molecules that support growth of heterotrophic bacteria
• Heterotrophic bacteria provide intrinsic stability and support growth of phototrophs by removing excess O2, increasing micro-nutrient availability, vitamin biosynthesis
• Algae-bacterial associations represent metabolically interactive, self-sustaining communities, which display adaptation to a range of harsh conditions
Ph
oto
au
totr
op
h
Hete
rotr
op
h
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Phototroph-Heterotroph Co-Cultures
Heterotrophic
bacterium
Phototroph
(microalga,
Cyanobacterium)
• Metabolic coupling: O2 produced by the algae is consumed by the heterotroph making stoichiometric amount of CO2 through oxidation of (endogenous or exogenous) organic C. Stoichiometric constraints drastically increase the intrinsic stability.
• Advantages:
-Improved mass transfer & productivity
- Increased range of carbon sources
- Modularity & ability to spatially separate the processes of light & CO2 capture with the downstream photosynthate conversion
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Coupling through Photosynthate Secretion
Glycogen
CO2
Photosynthesis
Glucose
O2
Other anabolism
ADPGluc Glucosyl-glycerol
Gluc6P +
UDPGluc
G3P +
Sucrose
Other carbohydrates?
Synechococcus sp. PCC 7002
ADPGluc
Cellulose
Rationale: Redirect fixed CO2 to mono/ disac-charide 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.
3C, 4C intermediates
NADH
Biomass, other respiration/fermentation
products
Sucrose Glucosylglycerol
In collaboration with Bryant’s Lab (Penn State)
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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.
Bryant, Xu et al., 2012 (in prep)
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- Heterotrophic growth supported through secretion of sugars and osmolytes (>300hs)
- Biomass concentration can be manipulated by varying growth conditions (light, CO2)
Metabolic Coupling through Secreted C
- Plug-and-play approach in which process of photosynthetic carbon fixation and product biosynthesis is spatially separated
Module A: CO2 -> Corg (sugars, organic acids)
Module B: Corg -> target bio-product
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Metabolic Coupling via Exogenous C
- Allows utilization of various C sources (including waste streams)
- Limited mass transfer as O2 and CO2 are produced throughout cultivation vessel
- Axenic (pure) cultures display significantly lower biomass productivity and growth rates
- Co-culture displays higher growth & productivity; does not need high mass transfer rates ; utilizes both carbon sources; no O2 accumulation
- Ratio of Corg/CO2 affects the proportion heterotroph & phototroph biomass
O2
CO2
Phototrophic
algae or cyanobacteria
Heterotrophic
bacterium
Solar
energy
Organic carbon
(waste)
Biomass,
value-added products
Co-culture (10 mM lactate, 5mM HCO3-, 50 rpm)
Heterotroph (10 mM lactate,
5mM HCO3-, 50 rpm)
Phototroph
(5mM HCO3-,
250 rpm) Phototroph
(5mM HCO3-,
50 rpm)
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Waste Treatment using Algal Co-cultures
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• Wastewater with high concentration of complex carbohydrates, N, and P
• Co-culture Bacilllus sp. and Haematococcus pluvialis
• Light, no bubbling, low agitation
Wastewater Treatment: Setup
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Wastewater Treatment: COD
100%
46%
29%
Untreated
wastewater
Treated
wastewater
Results after 200 hr incubation:
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Wastewater Treatment: Nitrogen
Untreated
wastewater
Treated
wastewater (10 days)
Results after 200 hr incubation:
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Production of High-Value Biomass
Biomass: 2.2 g/L
Algae: 1.4 g/L
Astaxanthin: ~ 0.8%
START END
Results after 200 hr incubation:
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Astaxanthin Accumulation
Value proposition:
- waste treatment (reduction in COD/BOD, N, P)
- high-value biomass production
- reduced mass-transfer, energy expenditures, as well as C emissions
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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.
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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 Burnham Inst. Medical Research: Dr. Andrei Osterman Dr. Jessica DeIngenis
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