This document summarizes research that evaluated the impact of varying concentrations of quaternary ammonium compounds (QACs), commonly used as sanitizers, on the structure and activity of microbial communities in thermophilic anaerobic digesters. The study found that QAC concentrations over 50 mg/L reduced methane production rates and altered the archaeal communities significantly, while bacterial communities were not significantly impacted. The results provide insight into specific QAC-sensitive archaea and how competition within archaeal communities changes at high QAC levels, decreasing digester performance.

1. Improving central carbon utilization pathways
for biofuel production
Outcomes
• Knocking out the competing phosphofructokinase I
(pfkA) promoted OPPP (~60% of glycolysis flux) and
the native EDP (~14% of glycolysis flux).
• Overexpression of edd and eda in the ΔpfkA mutant
directed ~70% of glycolytic flux through the EDP.
Background
• Hosts like Escherichia coli do not use their native
EDP for glucose metabolism even though this
pathway requires fewer enzymatic steps and is a
more thermodynamically favorable pathway than the
Embden–Meyerhof–Parnas pathway (EMPP) and
the oxidative pentose phosphate pathway (OPPP).
.
Significance
• E. coli was engineered to redistribute its native glycolytic flux and alleviate catabolite
repression.
• Our results revealed possible metabolic channeling, an overlooked mechanism by E. coli
to regulate its glucose catabolism and promote the hierarchy of substrate utilization for the
production of biofuels.
Hollinshead et al. (2016) “Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite
channeling using Δpfk mutants” Biotechnol Biofuels, 9, 212., doi, 10.1186/s13068-016-0630-y
DOE Office of Science
Graduate Student Research
(SCGSR) Program
Approach
• Disrupted phosphofructokinase I (pfkA) to promote
OPPP and the native EDP.
• Overexpressed edd and eda in the ΔpfkA mutant
• 13C-pulse experiments to measure the metabolite
labeling dynamics of sugar phosphates.

2. Mutation of the rice XA21 predicted nuclear
localization sequence does not affect
resistance to Xanthomonas oryzae pv. oryzae
Outcomes
• Ubi-XA21nls-GFP plants displayed slightly longer lesion
lengths, higher Xoo bacterial populations after inoculation
and lower levels of reactive oxygen species production
compared with the Ubi-XA21-GFP control plants.
Wei et al., (2016) “Mutation of the rice XA21 predicted nuclear localization sequence does not
affect resistance to Xanthomonas oryzae pv. oryzae” PeerJ 4:e2507 doi: 10.7717/peerj.2507
Background
• The rice XA21 receptor kinase confers robust resistance to
the bacterial pathogen Xanthomonas oryzae pv. oryzae
(Xoo).
• XA21 carries a predicted nuclear localization sequence
(NLS) that directs the C-terminal domain to the nucleus in
transient assays, whereas alanine substitutions in the NLS
disrupt the nuclear localization.
Significance
• Our findings demonstrate that the predicted NLS is not
critical for XA21-mediated immunity, an important finding in
the development of robust bioenergy crops.
Approach
• Determine if the predicted NLS is required for XA21-
mediated immunity using transgenic plants overexpressing
an XA21 variant carrying the NLS with the alanine
substitutions (Ubi-XA21nls-GFP).
Ubi-XA21nls-GFP plants display partial resistance to Xoo. (A) Leaves
of Kitaake, Ubi-XA21-GFP (XA21), and T2 progeny derived from three
independent Ubi-XA21nls-GFP T1 lines 14 days post-inoculation (dpi). (B)
Bacterial population in Kitaake, Ubi-XA21-GFP, and Ubi-XA21nls-GFP
plants at 0, 4, 8 and 12 dpi. (C) Lesion lengths of Kitaake, Ubi-XA21-GFP,
and Ubi-XA21nls-GFP plants at 14 dpi. The segregants (closed columns)
and null segregants (open columns) in each Ubi-XA21nls-GFP line were
separated into two groups for statistical analysis. Different letters indicate
significant differences between the groups (Tukey's HSD test, p < 0:05).

3. Rapid room temperature solubilization and
depolymerization of polymeric lignin at high
loadings
Outcomes
Achieved rapid room temperature solubilization of polymeric
lignin at high loading (31 wt%) in EG, which can be directly
applied for utilization of lignin. The solubilized lignin can be
easily and quantitatively recovered by using ethanol.
Theoretical and experimental studies indicate that role of H-
bond between EG and lignin molecules was critical for the
dissolution.
Sun et al. (2016) “Rapid room temperature solubilization and depolymerization of polymeric
lignin at high loadings” Green Chem., 2016, 18, 6012-6020 (Back cover paper) .
Background
The complex, highly cross-linked structure of lignin is a
major barrier that leads to its relatively poor solubility in most
solvents, which must be addressed in order to efficiently and
affordably valorize this biopolymer.
Significance
The present work provides an effective way to generate
highly concentrated lignin solution using relatively non-toxic
solvent, which can be further utilized for biochemical
conversion and biofuel applications.
Figure a: Up to 31 wt% polymeric lignin can be quickly
solubilized in EG at room temperature and the concentrated
lignin can be further depolymerized by hydrogen peroxide under
80 oC. Figure b: see Back cover image.
a
b
Approach
An inexpensive strong hydrogen bond (H-bond) donor solvent,
ethylene glycol (EG), was explored for weakening and/or
breaking down the interactions between lignin molecules at
room temperature. Computational and NMR characterization
were employed to gain mechanistic insights of H-bond
promoted lignin solubilization and valorization).

4. Catalytic transfer hydrogenolysis of ionic
liquid processed biorefinery lignin to phenolic
compounds
Outcomes
This process results in high liquid yields (65.5 wt%) with a
significant amount of monomers present (27 wt%) and
low char formation. Compositional analysis of the process
streams indicates that alkyl-substituted phenols are the
main products.
Kim et al. (2016) “Catalytic transfer hydrogenolysis of ionic liquid processed biorefinery
lignin to phenolic compounds” Green Chemistry, doi: 10.1039/C6GC02473D
Background
Valorization of residual lignin streams generated from
lignocellulosic biorefineries is key for economic viability
and sustainability.
Significance
This work suggests that residual lignin fractions from IL-
based lignocellulosic conversion technologies can be
depolymerized to value-added products and low
molecular weight platform chemicals for the renewable
fuels and chemicals sector.
Approach
Catalytic transfer hydrogenolysis using isopropyl alcohol
(IPA) as a hydrogen-donor solvent was employed at 300oC
to valorize lignin-enriched residues obtained from an ionic
liquid (IL) conversion process.
< Biorefinery concept of the integrated conversion process for whole biomass utilization >
Solid: 20 %
Gas: 15 %
Di-, tri- and oligomers: 50 %
monomers: 15 %
Liquids : 65 %
• Significant hydrogenated (saturated) monomeric phenols
• Residual lignin from IL-based lignocellulosic conversion can be depolymerized to value-
added products

5. Structure and activity of thermophilic
methanogenic microbial communities
exposed to quaternary ammonium sanitizer
Outcomes
QAC concentrations in excess of 50 mg/L were detrimental to
methane production in terms of production rate, apparent
yield, and methane quality. Archaeal sub-communities showed
significant increasing Bray-Curtis dissimilarity from the initial
QAC-free state as QAC levels increased. Conversely,
bacterial sub-communities did not significantly change.
Fernandez-Bayo et al. (2016) “Structure and activity of thermophilic methanogenic microbial communities exposed to quaternary
ammonium sanitizer” Journal of Environmental Sciences, doi: http://dx.doi.org/10.1016/j.jes.2016.10.005
Background
Quaternary ammonium compounds (QAC) are often used as
sanitizers in facilities that route wastewater to anaerobic
digesters. QACs may inhibit digester microorganisms and
affect biomethane output.
Significance
These data can inform best practices for operating
thermophilic anaerobic digesters. Furthermore, this study
provides novel insight into specific QAC-sensitive archaea
and elucidates competition among archaea at high QAC
levels that may relate to decreased digester performance.
Approach
Sludge from a thermophilic mixed organic waste anaerobic
digester was spiked with varying levels of QAC. Biogas
quantity and quality was subsequently monitored. 16S rRNA
gene sequencing was used to quantify changes in microbial
community structure in response to QAC level.
QAC levels in excess of 50 mg/L negatively impacted methane
production during thermophilic anaerobic digestion. Sludge archaea
underwent significant restructuring with increasing QAC concentration
while bacteria showed no significant restructuring.
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0 50 100 150
Bray-Curtisdissimilarityfrom
initialcommunity
QAC Level (mg l-1)
Archaea
Bacteria