JBEI Research Highlights - May 207

1. The OpenMSI arrayed analysis toolkit
Outcomes
• The software, called the OpenMSI Arrayed Analysis Tooklit (OMAAT) is
open-source and freely available at https://github.com/biorack/omaat
• OMAAT comes with Jupyter notebooks showcasing ways to use the
software in highly-automated as well as in more user-guided workflows
de Raad et al. (2017) Anal. Chem., doi: 10.1021/acs.analchem.6b05004
Background
• A promising new approach for high-throughput mass spectrometric
analysis is Mass Spectrometry Imaging (MSI) of arrayed samples,
allowing for the analysis of thousands of samples per day.
• A number of the JBEI Technology Division’s milestones are
contingent on the analysis of high-throughput Nanostructure-Initiator
Mass Spectrometry (NIMS) MSI data.
• High-throughput screening is a fledgling application of MSI, and no
automated analysis pipelines had been developed. In stead, the
picking of spots and the calculations have all been done manually.
Significance
• This software will allow researchers at JBEI, JGI and elsewhere
to analyze their arrayed-sample MSI data many times faster,
and enables the analysis of more thorough datasets.
Top: A typical MSI experimental workflow. OMAAT is used to locate
the spots in the sample array, and calculate their ion intensities.
Left: Illustration of OMAAT’s spot-finding algorithm
Right: In this dataset, relative ion intensities for each spot are a
measure of sugar concentrations.
Approach
• We developed a Python software toolkit automating the analysis of
arrayed samples in MSI datasets.
• We chose to use the existing OpenMSI software project
(https://openmsi.nersc.gov) as a data repository, avoiding the need
for users to transfer many-gigabyte multidimensional MSI data files.
• We integrated the software into a number of well-annotated Jupyter
Notebooks, allowing users to see appropriate instructions and
annotate their analysis pipelines.

2. The NADPH-oxidase AtRbohl plays a positive role in
drought-stress response in Arabidopsis thaliana
Outcomes
• Overexpression of RbohI in Arabidopsis significantly improves
drought tolerance.
• More H2O2 accumulated in RbohI overexpressors than in wild-type
plants in response to mannitol stress.
Background
• As the major resource of reactive oxygen species (ROS), the NADPH
oxidases (Rbohs) have been shown to play an important role in plant
cells under normal growth and stress conditions.
• Although many family members of Rbohs were studied, little is known
about the function of RbohI in Arabidopsis thaliana.
Significance
• This study provides insights on improving drought tolerance by
elevating H2O2 in plants. Improving drought tolerance will enable
bioenergy crops to be planted on some marginal lands in the US.
Approach
• The rbohI mutant and overexpressors were used to perform the
drought tolerance assay.
• The expression patterns were investigated using qRT-PCR.
• H2O2 contents in the RbohI overexpression lines and wild type
after treatment with mannitol stress were analyzed.
He et al. (2017) "The NADPH-oxidase AtRbohI plays a positive role in drought-stress response
in Arabidopsis thaliana". Biochem Biophys Res Commun. doi, 10.1016/j.bbrc.2017.05.131
1) Root growth and water loss of rbohI mutant in response to
drought stress in roots. (A) Lateral root length were measured at 10
days after transferring to different concentration of mannitol. (B) Water
loss in Col-0 and rbohI mutant plants.
2) Elevated drought tolerance in RbohI-YFP-overexpressing
plants. (A) Seedlings grown for 7 days on 1/2 MS medium were
transferred to 1/2 MS medium with or without 400 mM mannitol. (B)
Drought tolerance of 35S:RbohI:YFP (OE-3, OE-4 and OE-5) plants.
(C) H2O2 contents in the RbohI overexpression lines and Col-0 at 6
h after treatment with 400 mM mannitol.

3. Recent advances and challenges in
engineering efficient intracellular
cellobiose metabolism
Parisutham et al. (2017) “ Intracellular cellobiose metabolism and its applications in lignocellulose-based
biorefineries” Bioresource Technology, 239, 496-506, https://doi.org/10.1016/j.biortech.2017.05.001.
This review describes the recent
advances and challenges in engineering
efficient intracellular cellobiose
metabolism in industrial hosts. The
following points are discussed:
• Complete hydrolysis of cellulose is a
required aspect of biomass conversion
because the use of partial hydrolysis
products remains limited.
• Cellobiose, a cellodextrin, is the major
product of the enzymatic hydrolysis of
cellulose but is a partially hydrolyzed
product.
• Typical procedures convert cellobiose to
glucose with a β-glucosidase.
• An alternative to the complete extracellular
hydrolysis of celluloses can be envisioned
by engineering microbes with the ability to
hydrolyze and assimilate cellobiose.
• Microorganisms engineered to metabolize
cellobiose rather than the monomeric
glucose can provide several advantages for
lignocellulose-based biorefineries.
Three possible ways of cellulose utilization by industrial hosts: (i) complete
extracellular cellulose hydrolysis by cellulase cocktail; (ii) partial cellulose
hydrolysis and extracellular hydrolysis of cellobiose; (iii) partial cellulose hydrolysis
and intracellular assimilation of cellobiose.

4. 0
1
2
3
4
5
6
7
8
9
[E
O
A][O
Ac][D
EO
A][O
A
c][TEO
A][O
Ac]
[C
2C
1Im
][O
Ac]
[C
h][O
A
c]
[E
O
A
]F
[D
EO
A
]F
[TEO
A]F
[EO
A][La][TE
A][H
SO
4]
[EO
A
]C
l[E
O
A][H
S
O
4]
CalculatedvaluesofIonicliquids
Ionic liquids evaluated
Net Basicity
Acidity
Basicity
One-pot integrated biofuel production
using low-cost biocompatible protic ILs
Outcomes
• Low cost ethanolamine acetate ([EOA][OAc]) (~$1/kg) exhibits the
best comprehensive performance among the ILs investigated
• Without pH adjustment, water-wash and/or solid-liquid separation, the
whole pretreated slurry is directly used for ethanol production with
commercial enzyme cocktails and wild type yeast strains, generating
70% of theoretical yield (feedstock is switchgrass).
Sun et al. (2017) “One-pot integrated biofuel production using low-cost biocompatible protic ILs”,
Green Chemistry, DOI: 10.1039/C7GC01179B
Background
• Ionic liquid-based (ILs) “one-pot” process for biofuel production is still
challenging due to significant water-wash related to high toxicity of
[C2C1Im][OAc] and pH adjustment prior to saccharification for highly
basic [Ch][Lys]
• One-pot integrated biofuel production using low-cost biocompatible
protic ILs is highly desirable
Significance
• Discovery of low cost protic IL (e.g. [EOA][OAc]) enabled one pot
integrated process for biofuel production without pH adjustments,
water-wash and solid-liquid separations
Approach
• Synthesized 12 kinds of protic ILs, and predict their pretreatment
properties in terms of acidity and basicity by computational simulation.
• Screened their performance on lignin removal and sugar generations in
biomass pretreatment and saccharification processes
Low cost biocompatible ILs synthesis (A), evaluation
(B) and performance on glucose consumption and
ethanol production (C) in one-pot integrated process
Weight ratio
Biomass: H2O=1:27.5
Biomass: IL=1:1.5
Biomass: Yeast=33:1
Biomass: Enzyme=50:1
Switchgrass
Ionic liquid
Wild type yeast
Commercial enzyme
Switchgrass 1 kg
(0.329 kg glucose)
Glucan (R) 0.084 kg
Glucose (L) 0.003 kg
Ethanol (L) 0.117 kg
Ethanol yield 70%
H2O
Ethanol
Step 1
Step 2
A
B
C

5. Low cost ionic liquid-water mixtures for
effective extraction of carbohydrate and lipid
from algae
Outcomes
• Biomass derived from Chlorella vulgaris and Spirulina platensis can be
pretreated with low cost choline amino acid based ionic liquids to
effectively yield lipids (30.6% and 51% total lipids) and sugars (71% and
26% total sugars)
• Choline argininate yielded the most lipids and sugars out of all the ILs
studied
To et al. (2017). "Low cost ionic liquid-water mixtures for effective extraction of carbohydrate and
lipid from algae” Faraday Discuss. doi, 10.1039/c7fd00158d http://dx.doi.org/10.1039/C7FD00158D.
Background
• The most popular method to extract lipids from microalgae is Soxhlet
extraction using hexane, but this approach has several disadvantages
in terms of commercial viability
• This study explores the use of a series of cheap and environmentally
benign ionic liquids (ILs) for the pretreatment of microalgae. Made from
simple acid-base reactions between two naturally occurring non-toxic
chemicals, choline (an ammonium) and amino acids, these ILs are
significantly cheaper and less toxic than conventional ILs
Significance
• These results open new pathways towards dual production of
biodiesel and bioethanol from algae, using low cost ionic liquids
Approach
• Screened different types of inexpensive ionic liquids (ILs) for lipid
solubilization
• Process leaves behind a carbohydrate rich solid. The lipids were extracted
from the ILs with hexane, and the solid was subjected to enzyme hydrolysis
to release fermentable sugars
Distribution of lipids after pretreatment and
extraction. Blue bar: % lipids remaining in solid,
orange bar: % lipids extractable by hexane, grey bar:
% lipids remaining in liquor unextractable by hexane

6. High-quality genome sequences of four
lignocellulose-degrading bacteria from
Puerto Rican forest soil
Outcomes
• Genomes contain beta-ketoadipate pathway for aromatic
catabolism of lignin monomers and other phenolics into
tricarboxylic acid cycle intermediates
• Multiple dioxygenases, five different endo-1,4-betaxylanses
Woo et al. (2017) “High Quality Draft Genome Sequences of Four
Lignocellulose-degrading Bacteria from Puerto Rican Forest Soil- Gordonia
sp., Paenibacillus sp., Variovorax sp., and Vogesella sp.” ASM Genome
Announcements, 5:e00300-17. DOI: 10.1128/genomeA.00300-17
Background
• Lignocellulolytic bacteria are diverse in
enzymes and other protein machinery for
improving lignocellulosic biofuel production.
• Puerto Rican tropical forest soils were
targeted because the resident microbes
decompose biomass quickly and to near-
completion.
Significance
• These genomes are part of an ongoing investigation of the
genetic basis of lignocellulose degradation among bacteria
Approach
• Cellulose or lignin in minimal media was used to isolate these
organisms, with phenol oxidase, peroxidase, glucosidase,
cellobiohydrolase, xylopyranosidase, chitinase, CMCase, and
xylanase activities measured.
• Genomes of four isolates were high-quality draft sequenced.
Neighbor-joining
tree of bacterial
isolates with
hydrolytic
cellulase activity
(center bars) and
oxidative activity
(right-hand bars).

7. Parametric study for the optimization of
ionic liquid pretreatment of corn stover
Outcomes
• High sugar yields obtained at 50wt% biomass lading
after pretreatment at 140C, with glucose yields of
~87%
• [C2C1Im][OAc] can be employed at 90oC on milled
material with better results in terms of energy
efficiency than those obtained at 90oC with [Ch][Lys]
Papa et al. (2017) "Parametric study for the optimization of ionic liquid
pretreatment of corn stover” Bioresource Technology, 241, 627–637
Background
• JBEI has published several papers highlighting the
advantages of ionic liquid pretreatment
• The goal of this project was to conduct a wide survey of
experimental conditions and comparison of two different
leading ionic liquids to identify key parameters that are
critical in terms of process optimization
Significance
• This work provides a robust parametric dataset for further
studies on IL pretreatment scenarios that aim to reflect the
energy efficiency and cost drivers associated with ILs
Approach
• Parametric study of the efficacy of the ionic liquid (IL)
pretreatment (PT) of corn stover (CS) using 1-ethyl-3-
methylimidazolium acetate ([C2C1Im][OAc]) and cholinium
lysinate ([Ch][Lys]) was conducted. The glucose and
xylose released were generated from 32 conditions – 2
ionic liquids (ILs), 2 temperatures, 2 particle sizes (S), 2
solid loadings, and 2 enzyme loadings.
Energy consumption (MJ/Mg biomass) (bar charts) and (%) sugar
yield (marked statter) (of: unmilled and milled (2 mm) corn stover
pretreated with [C2C1Im][OAc] or [Ch][Lys] across different
pretreatment reaction conditions including temperatures of 140oC and
90oC, and solid loadings as Scenario A (50% w/w in pretreatment and
20% w/w for enzymatic hydrolysis) and Scenario B (15% w/w in
pretreatment and 6% w/w for enzymatic hydrolysis).
0
10
20
30
40
50
60
70
80
90
100
0
500
1000
1500
2000
2500
Unmilled 2 mm Unmilled 2 mm Unmilled 2 mm Unmilled 2 mm
[C2C1Im][OAc] [Ch][Lys] [C2C1Im][OAc] [Ch][Lys]
Scenario A Scenario B
%SugarYield
MJ/Mgbiomass
140°C, 5 mg/g glc.
140°C, 20 mg/g glc
90°C, 5 mg/g glc.
90°C, 20 mg/g glc.