November 2022 - Summary slides of Joint BioEnergy Institute (JBEI) research on biofuels and bioproducts.

1. Office of Biological and Environmental Research
Bioconversion of cellulose into bisabolene using Ruminococcus
flavefaciens and Rhodosporidium toruloides
Background/Objective
A proof-of-concept sequential bioreactor approach was investigated for
conversion of lignocellulose into biofuel.
Approach
• Organic acid fermentation was optimized for R. toruloides.
• Lignocellulose was anaerobically converted into organic acids by R.
flavefaciens, which were then aerobically converted into the jet fuel precursor
bisabolene by R. toruloides.
Results
• R. trouloides produced 8 g/L og bisabolene from a mix of organic acids.
• R. flavefaciens produced 4.2 g/L of organic acids, which were then converted
into 318 mg/L of bisabolene in a sequential bioreactor approach.
Significance/Impacts
This study exemplifies a consolidated approach for a multi-stage microbial
conversion of lignocellulose into biofuels.
Walls et. al. Bioresource Technology. doi: 10.1016/j.biortech.2022.128216)

2. Office of Biological and Environmental Research
Rapid quantification of alcohol production
in microorganisms based on NIMS
Background/Objective
• Primary alcohols with carbon chains ranging from C5 and C18 are
important biofuels and bioproducts targets.
• A bottleneck to improving the alcohols-producing strains is the lack
of a high throughput quantitative assay that has sufficient sensitivity
for direct characterization of 96-wells plate-based cell culture.
Approach
Developed a mass spectrometry-based high throughput assay using
TEMPO based oxidation followed by the formation of oxime-adducts,
which can be analyzed by NIMS
Results
This assay was successfully used to quantify C5 to C18 alcohols and we
applied this assay to gain new insights into P. Putida’s utilization of alcohols
and find that this strain largely could not grow on heptanol and octanol.
Significance/Impacts
This assay can rapidly quantify primary alcohol production
directly from cell cultures and enables quantitatively monitor both
alcohols as well as glucose and gluconate in the growth medium
Deng, K., et al. Analytical Biochemistry, DOI: 10.1016/j.ab.2022.114997
Fitness study of n-alcohols for Pseudomonas putida KT2440.
R OH R O
H
TEMPO, NCS
TBACl, CH2Cl2
dodecane
pH 8.6 buffer
O NIMS
H2N
STEP1 STEP2
R N
O NIMS
Oxime adducts
N
O
Me
Me
Me
Me
TEMPO
N O
O
Cl
NCS
(n-Bu)4N+Cl-
TBACl NIMS probe for aldehydes
F F
F F
F F
F F
F F
F F
F F
F
F
F
O
O
HN
H
N
NH
N
O
H
N
Me
Me
O
H2N
OH
C14
C16-1
C16
NCS + TEMPO oxidation
O NIMS
H2N
Oxime tagging
Me
OH
10
C12
Me
OH
12
13
OH
Me
13
N
C14
C16-1
C16
Me
N
10
C12
Me
N
12
13
N
Me
13
O NIMS
O
O
O
NIMS
NIMS
NIMS
O
C14
C16-1
C16
Me
O
10
C12
Me
O
12
13
O
Me
13
OH
C18-1
15
O
C18-1
15
N
C18-1
18 O NIMS
959
C14 fatty
alcohol
961
13
C-glucose
987
C12 fatty
alcohol
NCS + TEMPO
oxidation
Oxime tagging
C16-1-
ene fatty
alcohol
1013
C16 fatty
alcohol
1015
1041
C18-1-
ene fatty
alcohol
A B
m/z
A mass spectrometry-based assay to quantify primary
alcohols from cell culture

3. Office of Biological and Environmental Research
Precursor Prioritization for p-Cymene Production through
Synergistic Integration of Biology and Chemistry
Background/Objective
• p-Cymene is a promising molecule for the
production of fuels and chemicals
• Need to determine optimal biological target
Approach
• Compare the biological production of 1,8-
cineole and limonene
• Compare chemical conversion of each
Results
When considering the overall pathway, 1,8-
cineole is a better biological target as it has
lower toxicity and is equally readily converted to
p-cymene
Significance/Impacts
Demonstrates the need to evaluate the entire
synthesis pathway by considering both the
biological and chemical reactions
Lin, H.-H., et al., Biotechnol. Biofuels, DOI: 10.1186/s13068-022-02226-7

4. Office of Biological and Environmental Research
Background/Objective
• ClusterCAD is a reusable online database of natural polyketide synthase (PKS) “parts” to make small molecules
• Used successfully for a large number of PKS projects at JBEI and elsewhere
Approach
We expanded ClusterCAD to access greater chemical
diversity including ‘unusual’ PKSs and nonribosomal
peptide synthetases (NRPSs) that make amino acids.
Results
Users have over 7x as many natural parts, and better
search tools to design new enzymes that require fewer
(high-risk) engineering changes to create new molecules.
Significance/Impacts
We have lowered the barrier to access millions of
small molecules biologically with PKS/NRPS enzymes
that can make chemicals difficult or impossible with
other methods.
Tao, Xavier, et al. Nucleic Acids Research, https://doi.org/10.1093/nar/gkac1075
ClusterCAD 2.0: an updated computational platform
for chimeric type I PKS and NRPS design
https://clustercad.jbei.org

5. Office of Biological and Environmental Research
JBEI Enabled

6. Office of Biological and Environmental Research
Co-cultivation of anaerobic fungi with Clostridium
acetobutylicum bolsters butyrate and butanol production
from cellulose and lignocellulose
Background/Objective
Pairing C. acetobutylicum with an efficient lignocellulosic biomass degrader
like an anaerobic fungus provides an economic advantage by enabling
production of biobutanol and bio-butyrate from low-cost feedstocks
Approach
Construct and compare simultaneous and sequential co-culture strategies to
anaerobically produce butyrate and butanol from plant-derived biomass,
pairing C. acetobutylicum with multiple strains of anaerobic fungi
Results
• Higher levels of butyrate and butanol were produced by C. acetobutylicum in
co-culture with anaerobic fungi compared to monoculture controls
• RNA-Seq indicates that sequential cultivation may decrease time required to
produce butanol and relieve carbon catabolite repression
Significance/Impacts
Creating consortia that include these two microbes could be a
promising future avenue of industrial bio-butyrate and biobutanol
production from lignocellulosic feedstocks
Brown, J.L., et al. JIMB, https://doi.org/10.1093/jimb/kuac024

7. Office of Biological and Environmental Research
Environmental and Economic Impacts of Managing Nutrients in Digestate
Derived from Sewage Sludge and High-Strength Organic Waste
Background/Objective
Nutrient discharge from bioenergy facilities can pose risks to surrounding water
bodies, including aquatic life. Recovering nutrients prior to discharge can produce
valuable fertilizer products and avoid eutrophication impacts.
Approach
The study evaluates the potential for implementing nutrient recovery technologies at
bioenergy facilities, specifically using data from a local wastewater treatment plant.
Results
While nutrient recovery can dramatically reduce nitrogen loading in local water
bodies and reduce eutrophication impacts, the revenue from selling fertilizer do not
come close to offsetting the costs of installing and operating the systems.
Significance/Impacts
The study indicates that viable technologies exist for recovering nutrients from
wastewater generated through bioenergy production, but highlights the
challenges posed by high capital costs.
Orner, K.D., et al., Environmental Science & Technology. doi: 10.1021/acs.est.2c04020