Research summaries for papers published by the Joint BioEnergy Institute in February 2022

1. Modular automated bottom-up proteomic sample
preparation for high-throughput applications
Outcomes and Impacts
• Modular automated methods enable rapid, low
variance sample preparation of hundreds of samples
• We demonstrated its high reproducibility and
effectiveness for high-throughput proteomic analysis
on gram-negative bacteria and yeast samples (Fig. 2)
• We created digital protocols to facilitate methods
transfer and allow flexibility for future protocol
development.
Background
• High-throughput proteomic assays for
biotechnological and clinical applications require
automated solutions to minimize human error, save
time and resources, and improve data quality.
• Our fully-automated sample preparation method
lacks flexibility for novel organisms, different
amounts of cells, or other variations the entire
process.
Approach
• Modularize the proteomic sample preparation
procedure and automate the steps separately with
commonly-available liquid handlers (Fig. 1)
• Create detailed digital protocols to facilitate method
transfer and allow flexibility for future protocol
development.
Chen, et al., PLoS ONE. doi: 10.1371/journal.pone.0264467
Established digital protocol collection:
Fig 2.
Quantification of
>900 proteins
processed by the
modular automated
sample preparation
workflow on two
days shows
high reproducibility
(low coefficients of
variation (CV)).
Fig 1. Modular automated sample preparation
workflow for high-throughput proteomic assays.

2. Bioenergy Underground: Challenges and opportunities
for phenotyping roots and the microbiome for sustainable
bioenergy crop production
Background
• Bioenergy production often focuses on the aboveground biomass
for conversion to fuel and other materials. However, the
belowground component is crucial for soil carbon sequestration,
greenhouse gas fluxes, and ecosystem function. Roots maximize
feedstock production on marginal lands by acquiring nutrients and
water, and through interactions with the microbial community.
Approach
• This article provides a background on the need for belowground
research in bioenergy cropping systems, a primer on root system
properties of major U.S. bioenergy crops, and an overview of the
roles of root chemistry, exudation, and microbial interactions on
sustainability.
• The article was written jointly be representatives from all four BRCs
and other researchers at LBNL.
Outcomes and Impacts
• This review outlines recommendations and key challenges:
• Root phenotypes and soil microbiomes are critical for bioenergy crop
growth and sustainability, but are understudied
• Standardized methods and protocols should be developed and shared
through data sharing platforms
• Non-destructive phenotyping tools should be developed
• Machine learning and artificial intelligence can play an important role in
understanding the complex correlations and spatiotemporal patterns in
root and soil systems.
Dominant traits that should be optimized belowground in
bioenergy crops include those affecting root system
architecture, chemistry and exudation, nutrient transport,
and the microbiome. These belowground optimizations will
lead to CO2 capture, increased production and income, and
improved soil health and carbon sequestration
York et al. (2022) The Plant Phenome Journal. 5: e20028. DOI: 10.1002/ppj2.20028

3. Sustainable manufacturing with synthetic biology
Background
• Synthetic biology promises to lead the way to a
sustainable manufacturing sector. If the
thousands of chemicals derived from petroleum
and natural gas — including fuels, plastics and
industrial chemicals — could be produced
instead with microbes, annual savings in global
GHG emissions would be substantial.
Approach
• We comment on a recent work by Liew et al.
published in Nature Biotechnology and provide
broader context for the role of synthetic biology
in decarbonizing the industrial sector.
Outcomes and Impacts
• The contribution of synthetic biology to creating
a sustainable manufacturing sector would be
greatly enhanced though diversification of the
organic feedstocks that microbial hosts can
convert.
• If all non-fuel petrochemicals were produced
instead through synthetic biology, about 0.5 Gt
of CO2 globally each year could be avoided
• Creating a circular or even net-negative
industrial sector through novel synthetic biology
approaches will not solve the climate crisis on
its own, but it can tackle some of the most
difficult-to-decarbonize parts of the global
economy.
Scown & Keasling (2022) Nature Biotechnology, doi: 10.1038/s41587-022-01248-8
Methods for engineering the acetogen Clostridium autoethanogenum to
convert industrial waste gases into useful industrial chemicals at
commercially relevant efficiency, selectivity and scale

4. JBEI Enabled

5. Making Security Viral: Shifting Engineering Biology
Culture and Publishing
Background
• Constructing, synthesizing, and editing genes and genomes rapidly at scale has far-reaching implications for society
• As SARS-CoV-2 spread in early 2020, researchers rapidly mobilized. Knowledge sharing was crucial to making progress
• Several publications described the de novo construction of SARS-CoV-2 in the laboratory, one in the form of a protocol
• Given the demonstrable harm caused by the virus, the unequal distribution of mitigating vaccines and therapeutics, their
unknown efficacy against variants, and the interest in this research by laboratories unaccustomed to working with highly
transmissible pandemic pathogens, there are risks associated with such publications, particularly as protocols
Approach
• We describe considerations and offer suggestions for enhancing security in the publication of synthetic biology research
• We recommend:
(1) that protocol manuscripts for the de novo synthesis of certain pathogenic viruses undergo a mandatory safety
and security review;
(2) that if published, such papers include descriptions of the discussions or review processes that occurred
regarding security considerations in the main text; and
(3) the development of a governance framework for the inclusion of basic security screening during the publication
process of engineering biology/synthetic biology manuscripts to build and support a safe and secure research
enterprise that is able to maximize its positive impacts and minimize any negative outcomes
Outcomes and Impacts
• This paper’s recommendations are geared to journals that individually, or in concert, can take steps to increase security
• A successful end state might have parallels with the practices of DNA synthesis providers
• Similarly, government guidance could help journals implement appropriate publication standards for manuscripts with
potential security concerns
• Implementing these changes will support the development of a stronger culture of security in engineering biology
research and publication
• Funders can also encourage a culture of security by requesting that proposers include precautions and/or mitigation
strategies for work with security implications, and by tracking potential risks through the project lifecycle via progress
reports for funded projects
Mackelprang et al. (2022) ACS Synthetic Biology doi: 10.1021/acssynbio.1c00324

6. Economic and greenhouse gas analysis of regional
bioenergy-powered district energy systems in
California
Background
• Biomass feedstocks and residual organic waste from biorefineries
can serve as useful fuel in thermal energy generation
• There is an urgent need for building energy services that are not
only low-carbon but reliable. District energy systems (DES) have
the potential to meet building power, heating and cooling needs
efficiently and reliably, but further research is needed to understand
their coupling with local renewable energy sources such as
biomass.
Approach
• We assess the cost and greenhouse gas implications of using
bioenergy from local organic waste to power DES in California
communities. We describe a set of possible scenarios for
bioenergy integration into DES, including fuel switching and
retrofitting of existing systems and DES expansion through new
projects
Outcomes and Impacts
• New bioenergy-powered district energy systems have higher
revenue from energy sales and higher incurred costs than
incumbent fossil fuel powered building thermal systems.
• Switching from natural gas to RNG in existing district energy
systems may result in cost savings.
• The costs and benefits of district energy systems need to be more
clearly communicated to accelerate adoption and integration.
• Bioenergy-powered district energy systems can offer a cost of
carbon abatement on par with distributed solar-powered building
systems.
Breunig et al. (2022) Resources, Conservation & Recycling, doi: 10.1016/j.resconrec.2022.106187
Top: Potential configurations for district energy
systems utilizing bioenergy
Bottom: Amortized annual cost of district energy
systems and non-district energy

7. Lower-Cost, Lower-Carbon Production of
Circular Polydiketoenamine Plastics
Background
• The efficiency by which monomers may be recovered during the
chemical recycling of plastic waste has thus far dominated the
discussion over which future polymer chemistries might be more
sustainable than those in use today.
• At scale, other factors emerge as equally important, such as the
costs of primary versus secondary resin production as well as the
energy and carbon intensity of circular manufacturing processes.
Approach
• We apply systems analysis to identify problematic chemical
processes used for the primary production of plastics designed for
infinite recyclability: polydiketoenamine (PDK) resins from novel
triketone and amine monomers.
Outcomes and Impacts
• We advance a less intensive process for triketone production,
which lowers the cost of primary PDK production by 57% and
results in 66% less life-cycle greenhouse gas (GHG) emissions.
• Using the automotive sector as a case study, we discuss the
impact of replacing nonrecyclable polyurethane with circular PDK
over the next 60 years. We find that the cumulative GHG
emissions associated with introducing PDK are half those of
staying the course with polyurethane.
• Introducing bio-based components to PDK will be key to reducing
the GHG emissions and moving toward a circular plastics economy
Demarteau et al. (2022) ACS Sustainable Chemistry & Engineering, doi: 10.1021/acssuschemeng.1c07851
Top: Minimum selling price of PDK resins
Bottom: Life-cycle GHG footprint of PDK resins