Biological Conversion of Glycerol:

A New Path to Renewable Chemicals

and Fuels
Ramon Gonzalez
Department of Chemical & Biomolecular Engineering,
Department of Bioengineering,
Rice University
 Renewable Chemicals and Fuels
Linking PetroChemical and BioBased Industries

Petrochemical Industry Biobased Industry

Crude oil and natural gas Plant biomass and wastes

Hydrocarbons Sugars, oils, etc.

• National energy security
• Climate protection
Feedstocks and intermediates • Sustainability
• Economic growth
Fuels: Biofuels:
Gasoline, diesel Fuels Ethanol, biodiesel
Chemicals Bulk and Specialty Chemicals Chemicals
Materials Bio-based Materials
 Fuels & Chemicals from Biorenewables
Challenges

Feedstock Feedstock Microbial Recovery

Engineering Deconstruction Fermentation Distillation
Extraction
Plant Pretreatment and Biocatalysis
Biotechnology Hydrolysis
 Metabolic Engineering (MetEng)
and Metabolic Evolution (MetEvo)

STRAIN CONSTRUCTION
Genetic engineering (gene/pathways
“knockins” & “knockouts”);
Mutagenesis & Selection

STRAIN
CHARACTERIZATION
Tubes, Flask &
MetEng/MetEvo Bioreactors
DESIGN
Integration Cycle
(systems biology),
Target
Identification
ANALYSIS
Fermentation Profiling,
Functional genomics

Fluxomics

Proteomics
Transcriptomics

Metrics: Concentrations (g/L), Yields (g/g), Rates (g/L/h)

 Oleochemical and Biodiesel Industries
Glycerol/Glycerin as Inevitable Byproduct

Yazdani and Gonzalez (2007). Curr. Opin

Biotechnol. 18: 213-219.
Biodiesel and Crude Glycerol
US Production & Prices

Yazdani and Gonzalez (2007) Curr. Opin Biotechnol. 18: 213-219.

 Solution?

The Use of Anaerobic Fermentation of

Glycerol as a Platform to Produce
Reduced Chemicals and Fuels

Yazdani and Gonzalez (2007) Curr. Opin Biotechnol. 18: 213-219.

 Why Anaerobic Fermentation
of Glycerol?
Relative and Absolute Reasons
• Abundant: The production of biodiesel in the U.S.
increased by 10-fold in the last two years: in million
gallons, from 25 (2004) to 250 (2006). Each gallon of
biodiesel inevitably generates 0.75 lb of glycerol.
• Inexpensive.
• Renewable/Green.
• New Uses/Applications Needed.
• Lower Capital/Operational Costs of Anaerobic
Fermentations.
• Highly Reduced State of Carbon (outperform sugars:
see next slide).
 Glycerol vs Sugars
Redox Balance

Dharmadi, Murarka, Gonzalez. Biotechnol. Bioeng., 2006: 94: 821-829. Gonzalez et al. (MS in Review);
Murarka et al. App. Environ. Microbiol., 2008: 74 (4); Yazdani & Gonzalez. (2007) Curr. Opin. Biotechnol. 18: 213-219.
Current State of Knowledge on Glycerol
Metabolism in Enterobacteriaceae
Established model for glycerol
fermentation: Glycerol-fermenting
species such as Klebsiella and
Citrobacter utilize a two-branch pathway;
the reductive, 1,3-PDO-producing branch
acts as a sink for the reducing equivalents
generated in the oxidative branch.

Respiratory metabolism of
glycerol: Metabolism of glycerol
in species such as E. coli, which do
not synthesize 1,3-PDO, takes place
through a respiratory pathway that
requires an external electron
acceptor.
Gonzalez et al. (MS in Review); Murarka et al. App. Environ. Microbiol., 2008: 74 (4);
Yazdani & Gonzalez. (2007) Curr. Opin. Biotechnol. 18: 213-219.
 Our Findings: E. coli Ferments Glycerol
in the Absence of Electron Acceptors
For nearly 80 years,
researchers had believed that Glycerol
the metabolism of glycerol in
E. coli was restricted to
respiratory conditions: i.e.,
the presence of an external
electron acceptor.
Growth
• Quastel JH, Stephenson M µ =0.041 h-1
(1925). Biochem J. 19:660.
• Quastel JH, Stephenson M,
Whetham MD (1925). Biochem Ethanol
J. 14:304.
• Lin EC (1976). Annu Rev
Microbiol. 30:535-78. Other Products
• Booth I (2005). In Neidhardt
FC et al., (ed.), E. coli and
Salmonella: cellular and
molecular biology, Web
Edition. ASM Press,
Washington, DC. Available at
Dharmadi, Murarka, Gonzalez. Biotechnol. Bioeng., 2006: 94: 821-829.
www.ecosal.org. Gonzalez et al. (MS in Review); Murarka et al. App. Environ. Microbiol., 2008: 74 (4);
Yazdani & Gonzalez. (2007) Curr. Opin. Biotechnol. 18: 213-219.
 Can we Change the Biodiesel Industry
with Glycerol Fermentation?
Feedstock and Operating Costs for B100
Component/Kind of Cost Cost in $/gal. biodiesel (12/06)
Soy Oil 2.381
Methanol 0.202
Processing Costs 0.211
(Feedstock + Operating) Costs 2.794

B100 Market Price 2.750

Byproduct (Glycerin) Credit 0.021 (@0.025 $/lb)

“Byproduct Ethanol-H2/Formic credit” 0.150-0.250

“Byproduct Succinic Acid Credit” 0.600-1.000
 Co-production of Ethanol-Formic
Strain SY04 (pZSKLMgldA)

Glycerol Growth

EtOH

Formic

Byproducts

Product yields: ~ 95% (mol EtOH or formate/mol glycerol)

Specific Rates: ~ 30 mmol product/gCell/h
Yazdani and Gonzalez (MS in Review)
 Co-Production of:
(1) EtOH-Formic or (2) EtOH-H2

Data represents close to

maximum theoretical
yield for all products

SY03 (pZSKLMgldA) Strain 2 SY04 (pZSKLMgldA)

Formate or Hydrogen can never be co-produced along with ethanol if sugars

are used instead of glycerol. The use of glycerol results in more energy- and
carbon-efficient processes.
Dharmadi, Murarka, Gonzalez. Biotechnol. Bioeng., 2006: 94: 821-829.
Gonzalez et al. (MS in Review); Murarka et al. App. Environ. Microbiol., 2008: 74 (4);
Yazdani & Gonzalez. (2007) Curr. Opin. Biotechnol. 18: 213-219.
Ethanol-Formic and Ethanol-H2
from Glycerol
* Feedstock cost is
“net” for corn-
ethanol (include
revenue from co-
products) but not
for glycerol-derived
ethanol (does not
include revenue
from co-products H2
and formic acid)

Capital cost
significantly lower
than in corn-ethanol
plant.

Yazdani and Gonzalez (2007) Curr. Opin Biotechnol. 18: 213-219.

 Can we Change the Biodiesel Industry
with Glycerol Fermentation?
Feedstock and Operating Costs for B100
Component/Kind of Cost Cost in $/gal. biodiesel (12/06)
Soy Oil 2.381
Methanol 0.202
Processing Costs 0.211
(Feedstock + Operating) Costs 2.794

B100 Market Price 2.750

Byproduct (Glycerin) Credit 0.021 (@0.025 $/lb)

“Byproduct Ethanol-H2/Formic credit” 0.150-0.250

“Byproduct Succinic Acid Credit” 0.600-1.000
 Acknowledgements
Graduate Students: Postdoc:
Y. Dharmadi A. Murarka S. Yazdani
A. Gupta J. Clomburg
G. Durnin

USDA - National Research Initiative of the USDA Cooperative

State Research, Education and Extension Service (2005-35504-
15222/16698)
NSF- Division of Chemical, Bioengineering,
Environmental, and Transport Systems
(CBET-0601549)