Lawrence Livermore National Laboratory



March 2019


New Publication on how beetle gut microbes make food and fuel

A beetle

Photo courtesy of Javier A Ceja-Navarro

When most people think of wood eating insects, they imagine termites that can destroy a home or business.

However, as part of a Lawrence Livermore National Laboratory bioenergy study, project scientist Jennifer Pett-Ridge and collaborators have learned how the digestive system of a wood-eating beetle serves as a natural mini-reactor for biofuel production.

Led by Javier A. Ceja-Navarro from Lawrence Berkeley National Laboratory (LBL), the team looked at the microorganisms living within the digestive tract of ‘Horned passalus’ beetles (Odontotaenius disjunctus), which live in fallen logs (harvested in Louisiana from fallen pecan tree logs) and are “subsocial”—living in colonies within the wood. These beetles, which are common throughout the Eastern U.S., are responsible for decomposing a huge amount of wood and transforming it into soil organic matter, which contributes to the health and nutrient stock in forest soils. The fact that these beetles can live on food (wood) that is very poor quality—comprised mainly of lignin, cellulose, and hemicellulose with very little nitrogen—is thought to be linked to the capabilities of the bacteria and fungi that reside within their digestive system.

“Understanding how the gut microbiome populations interact to deconstruct lignocellulosic materials to sugars or potential biofuels such as hydrogen and methane could potentially aid in the optimization of industrial cellulosic degradation,” said LLNL biogeochemist Jennifer Pett-Ridge, a co-author on a paper appearing in the journal Nature Microbiology.

The beetle processes large amounts of woody biomass and has developed symbiotic relationships with gut microbes to survive on a low-nutrient diet. The microbial community in the beetle’s gut provides digestive enzymes that degrade complex polysaccharides and lignin of plant cells. Physically, the gut is highly compartmentalized, resulting in a segmentation of microbial populations and the processes for energy extraction from lignocellulose.

To understand the complex connections that make up the O. disjunctus digestive system, the team defined the environment in which these organisms interact, identified the location of key species and their genomes, and confirmed the metabolic pathways contributing to their efficient metabolism of lignocellulosic material.

Breaking down lignin and cellulose to make fuel (sugars etc.) is chemically very difficult, as is fermenting it into a useable reduced product (like acetate, methane or hydrogen). The beetle can’t do this on its own. It needs a diverse group of bacteria and other microbes in its gut to help. The spatial stratification in the beetle’s gut allows for different microenvironments—some are more acidic, some more anaerobic. Each appears to have a community that sequentially degrades the wood substrates and extracts energy, sharing some products with the beetle host.

“We study them because they are a natural model biorefinery,” Pett-Ridge said. “Understanding how evolution has solved the complex lignocellulose to fuel conversation process can help us design better industrial mimics and find novel enzymes or pathways.”

Coarse woody debris makes up a tremendous amount of biomass in forest ecosystems. As a result, several insect groups have evolved to take advantage of this abundant resource, forming specialized wood-feeding guilds. Among the most prominent groups of insects that have specialized in this manner are termites and specific groups of beetles. These groups are considered economically important insects due to the destructive character of some of their members, as examples, subterranean termites, powderpost beetles, bark beetles and longhorn beetles. These insects are able to perform mechanical and enzymatic breakdown of coarse woody biomass that is critically important to the ecology of these ecosystems. This ability to subsist on woody biomass, is in part due to the symbiotic associations that have evolved between these insects and their gut microorganisms.

“These passalid beetle gut communities represent an excellent model system for determining how microbial communities assemble and partition processes along physicochemical gradients to yield an efficient system that converts lignocelluloses into hydrogen, methane, ethanol and other bioenergy-relevant products,” Pett-Ridge said.

Other institutions contributing the research include University of California, Berkeley, Purdue University, Louisiana State University, University of South Carolina, Pacific Northwest National Laboratory and Oregon State University.

The work was funded by the Department of Energy’s Office of Science.

For more information, read the article in The Daily Californian.

[Ceja-Navarro JA, Karaoz U, Bill M, Hao Z, White RA, Arellano A et al (2019). Gut anatomical properties and microbial functional assembly promote lignocellulose deconstruction and colony subsistence of a wood-feeding beetle. Nature Microbiology.]



SFA team member chairs session on photoheterotrophy

Photoautotrophy-dominated environments, such as natural ecosystems like lake, river, and ocean surfaces, as well as engineered ecosystems like photobioreactors, algal turf-scrubbers, and outdoor algal raceways, support entire ecosystems fueled by photosynthesis and dominated by heterotrophic bacterial processes feeding on this recently-fixed carbon. Many heterotrophic bacteria in these ecosystems harbor the genetic machinery to harvest light energy themselves, a process called photoheterotrophy. SFA team member Xavier Mayali recently co-chaired a session at the American Society of Limnology and Oceanography annual meeting in February (with Laura Gomez-Consarnau from the Centro de Investigación Científica y de Educación Superior de Ensenada, Baja California, Mexico) to bring together experts in this growing area of research. Recent discoveries on this topic presented in the session include a newly-discovered type of rhodopsin and quantification of total energy absorbed by photoheterotrophic processes compared to chlorophyll, which further suggest photoheterotrophy is a major global process and may also be important in algal raceway ponds.


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February 2019


New Publication on bacterial genome annotation

Comparison chart

Comparison of reactions identified for single versus combined annotation sources in four SFA bacterial isolate genomes.

Genome-scale metabolic modeling is a cornerstone of systems biology analysis of microbial organisms and communities, yet these genome-scale modeling efforts are invariably based on incomplete functional annotations. Annotated genomes typically contain 30–50% of genes without functional annotation, severely limiting our knowledge of the “parts lists” that the organisms have at their disposal. In our recent publication, we present results on a comprehensive reannotation of 27 bacterial reference genomes, focusing on enzymes with EC numbers annotated by KEGG, RAST, EFICAz, and the BRENDA enzyme database, and on membrane transport annotations by TransportDB, KEGG, and RAST. Our analysis shows that annotation using multiple tools can result in a drastically larger metabolic network reconstruction, adding on average 40% more EC numbers, 3–8 times more substrate-specific transporters, and 37% more metabolic genes. These results are even more pronounced for pathways outside of the core carbon metabolism, and for bacterial species that are more phylogenetically distant from well-studied model organisms such as E. coli. Our team is currently developing tools for the DOE Systems Biology Knowledgebase to allow users to upload functional annotations from popular third-party annotation tools, compare and merge them, and use them for metabolic modeling.

[Griesemer M, Kimbrel JA, Zhou CE, Navid A, D’haeseleer P, Combining multiple functional annotation tools increases coverage of metabolic annotation, BMC genomics 19 (948), 2018.]


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November 2018


Welcoming two new Lawrence Scholar graduate students to LLNL

Colleen Hui Katie Harding

New Lawrence Scholars Colleen Hui (left) and Katie Harding (right)

SFA team members are hosting two new Lawrence Scholar graduate students.

Colleen Hui is joining us from Sabeeha Merchants group at UC Los Angeles/UC Berkeley. Her research has centered on understanding iron homeostasis in the unicellular green alga Chlamydomonas reinhardtii. At LLNL she will work with NanoSIMS lead Peter Weber on imaging intracellular trace metal metabolism in Chlamydomonas. Specifically, she will be using LLNL’s unique NanoSIMS capability to 1) distinguish the iron storage site in Chlamydomonas cells, 2) analyze the dynamics of intracellular iron movement, and 3) investigate the roles of three candidate vacuole-associated proteins (NRAMP4, CVL1, CVL2) in relation to iron storage and transport in Chlamydomonas. The outcome of this project will provide a better understanding on trace metal utilization in a model alga for sustainable biofuel production.

Katie Harding, a graduate student in Jon Zehr’s group at UC Santa Cruz, is joining the SFA group as a part-time Livermore scholar. Her thesis research is focused on marine nitrogen fixation by unicellular bacteria. At LLNL she will work with Xavier Mayali on using geneFISH (fluorescent in situ hybridization for functional genes) to link microbial functional gene presence and biological activity using NanoSIMS and isotope probing.



SFA team member presents at 2018 International Conference on Microbiome Engineering

An example of a computational model

SFA member Ali Navid attended the 2018 International Conference of Microbiome Engineering in Boston, MA, in November to present some of his work on computational modeling of microbe-algae and microbe-plant interactions. He presented a talk entitled “Examination of metabolic interactions between phototrophs and their symbiotic microbiome” in the session “From genome-scale to community-scale models.” Learn more about the conference by visiting the 2018 International Conference of Microbiome Engineering website.


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October 2018


New Publication on niche partitioning in microbial mats identified using metagenomic analyses

PCA galaxy chart with MG-RAST annotated abundant phyla labeled (A) and final detected bins.

In this study, we examined hypersaline microbial mat communities, which are known for their taxonomic and metabolic diversity and steep environmental gradients. Here, we illustrate how metagenome sequencing can be used to meaningfully assess microbial ecology and genetic partitioning in these complex communities, using binning to reconstruct organisms in silico to assess ecosystem partitioning. We were able to distinguish putative core and accessory genes for the dominant Cyanobacteria in the system, Coleofasciculus chthonoplastes, indicating highest differentiation in nutrient utilization and stress response, suggesting salinity, metals, and light may drive differentiation in this group. We also identified a distinct set of glycoside hydrolases in C.chthonoplastes, building on previous SFA research showing the importance of these polysaccharides in carbon cycling in the mats. The analysis also uncovered evidence of putative phototrophs within the Gemmatimonadetes and Gammaproteobacteria.

[Lee, JZ, Everroad, CR, Karaoz, U, Detweiler, AM, Pett-Ridge, J, Weber PK, Prufert-Bebout, L, Bebout, BM. Metagenomics reveals niche partitioning within the phototrophic zone of a microbial mat. PLoS ONE 13(9): e0202792. 2018]



Collaboration to study sustainable bioenergy agriculture

the people involved with the collaboration

The LLNL Biofuels SFA is collaborating with Dr. Christine Hawkes (NC State) to improve our understanding of sustainable bioenergy agriculture—studying plant symbionts and belowground processes such as water use efficiency and soil C storage. In a recent experiment, LLNL post-college appointee Max Li worked with the Hawkes lab to harvest Panicum virgatum plants from a study of how foliar fungal endophytes affect plant drought physiology. These endophytes range from antagonistic to beneficial, and are expected to affect the plant’s physiology, biomass, and root metabolites/gene expression.


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August 2018


New Publication on carbon fixation enhancement in two species of microalgae by attached microbiomes

NanoSIMS image of a P. tricornutum and heterotrophic bacteria co-culture. The left panel shows the microalgae enriched in freshly fixed carbon, the middle panel shows the varied morphologies of active bacteria comprising the microbiome, and the right panel is a merged view.

We observed mutualistic interactions between heterotrophic bacteria and two species of biofuels-relevant microalgae, Nannochloropsis salina and Phaeodactylum tricornutum, mediated by physical association between individual cells. At the bulk scale, microalgae in these co-cultures exhibited enhanced growth and yield. At the microscale, we used the LLNL NanoSIMS to observe that both species exhibited enhanced carbon fixation in response to the presence of the microbiomes, but there were divergent responses by each species to bacterial attachment. We illustrate how P. tricornutum may be predisposed to interact mutualistically with bacteria via attachment, but N. salina does not share these traits. Attached bacteria benefit from these relationships by receiving more reduced carbon from their algal host compared to free living cells.

[Samo TJ, Kimbrel JA, Nilson DJ, Pett-Ridge J, Weber PK, Mayali X. Attachment between heterotrophic bacteria and microalgae influences symbiotic microscale interactions. Environmental Microbiology 2018, doi: 10.1111/1462‐2920.14357]


Master’s thesis defense

Chorazyczewski and his thesis advisor

SFA team member Adam Chorazyczewski, a master’s student with Dr. Paul Zimba at Texas A&M successfully defended his master’s thesis entitled “Do phycosphere-associated bacteria affect the growth and lipid accumulation of Phaeodactylum tricornutum.” His SFA-funded research involved profiling and characterizing growth and lipid accumulation in co-cultures of P. tricornutum and 16 separate bacterial species provided by LLNL team members. He identified bacterial isolates with positive effects on both growth and single cell lipid content. SFA algae-bacterial lead Xavier Mayali served as a committee member.




EMSL proposal awarded

SFA team members led by Ty Samo were awarded an EMSL grant entitled “Nano- to microscale characterization of metabolic cooperation facilitated by physical associations between phototrophic microalgae and heterotrophic bacterial symbionts.” Co-Investigators include R. Stuart, X. Mayali, C. Ward, P. Weber, T. Northen (LBL), and C. Buie (MIT).


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June 2018


Algae Outreach Event at LLNL

SFA team members Carolyn Fisher (Sandia), Xavier Mayali (LLNL), and Chris Ward (LLNL) helped host a hands-on work station, called “Algae Invader Investigation,” on algae and algal predators. Students learned about bioenergy research on algae and the current cultivation obstacles, such as algal predators. They used both microscopes and household items such as beans and pasta to demonstrate how algae are studied in the lab. According to the Sandia summer intern Franny Carcellar: “I had one girl tell me after looking through the microscope that now she wants to be a scientist. If we just inspire one kid, then it’s all worth it!”

For more information on the event, see the article titled “STEM Day at the Lab gives underserved students a taste of the wonders of science” on the LLNL website.


SFA team member Xavier Mayali (left) showing a student how to use the microscope to look at algae and their predators.

Algae Invader Investigation Team from left to right: Franny Carcellar, Colleen Courtney, Annette LaBauve, Carolyn Fisher, Raga Krishnakumar, Chris Ward, and Xavier Mayali.

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May 2018


New publication on multimodal LA-ICP-MS and nanoSIMS imaging

NanoSIMS image of copper and phosphorus signals in the zebrafish embryo retina, showing copper is enriched in puncta in the outer nuclear layer

NanoSIMS image of copper and phosphorus signals in the zebrafish embryo retina, showing copper is enriched in puncta in the outer nuclear layer.

We are developing high sensitivity, high spatial resolution methods to image essential metals in biological systems. Here, we collaborated with the C. Chang Lab at UC Berkeley to standardize our NanoSIMS copper measurements and apply the method to analysis of copper metabolism in zebrafish. Using this method, we were able to demonstrate that the fish metal distribution system prioritizes delivering copper to the eye, despite a severe copper deficit caused by a genetic mutation which mimics a human copper dysregulation disorder, Menkes disease.

[Ackerman CM, Weber PK, Xiao T, Thai B, Kuo TJ, Zhang E, Pett-Ridge J, Chang CJ. Multimodal LA-ICP-MS and nanoSIMS imaging enables copper mapping within photoreceptor megamitochondria in a zebrafish model of Menkes disease. Metallomics 2018 10(3):474-85.]



Greenhouse at LLNL now open!

The LLNL greenhouse

Our new greenhouse facility is now operational. It is a new IGC Arch Series 6500 greenhouse (1800 sf) with full temperature and light controls. It will eventually house an array of Coy isotope labeling chambers for plant 13CO2 isotope labeling and our Picarro portable Cavity Ring-Down Spectroscopy (CRDS) analyzer. We are currently setting up a series of plant-AMF inoculations.




Invited seminar on computational systems biology research

Presenter with his powerpoint slide on the wall behind him.

SFA team member Ali Navid recently gave an invited seminar as part of the LLNL’s and nearby Las Positas College student science and engineering seminar series. His talk, given to a large audience of community college science students and faculty, was titled “Computational Systems Biology: Simulating life from microbes to humans.” He discussed the state of computational systems biology in general and particularly at LLNL and presented examples of different types of modeling (e.g., constraint-based genome-scale, dynamic pharmacology, and 3D microbial biophysics models) that span multiple time and spatial scales from his SFA and biosecurity-related research.






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April 2018


New Publication: Quantitative isotope incorporation reveals substrate partitioning in a coastal microbial community

Taxon-specific Chip-SIP isotope incorporation data mapped onto the degradation pathway of various phytoplankton-derived organic substrates.

Taxon-specific Chip-SIP isotope incorporation data mapped onto the degradation pathway of various phytoplankton-derived organic substrates

SFA team members Xavier Mayali and Peter Weber have a new publication, supported by the Biofuels SFA, on the use of the Chip-SIP method (NanoSIMS and microarrays) to quantify the taxon-specific incorporation of algal-derived organic components by bacteria and eukaryotes. They carried out simultaneous incubations with 14 different stable isotope labeled substrates to examine phylogenetic signal of resource utilization and mixotrophy. The unique aspect of examining such a high number of substrates enabled them to identify substrate partitioning, and the data showed that two thirds of the taxa exhibited unique incorporation patterns, with strategies ranging from generalists to specialists.

[X. Mayali and P.K. Weber, Quantitative isotope incorporation reveals substrate partitioning in a coastal microbial community, FEMS Micro Ecol 94 (5), fiy047 (2018), doi:10.1093/femsec/fiy047]


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March 2018


SFA team members attend annual JGI user meeting

Xavier Mayali accepts the “Outside the Box Poster Award” from JGI Director Nigel Mouncey.

Xavier Mayali accepts the “Outside the Box Poster Award” from JGI Director Nigel Mouncey.

Five SFA team members presented at the 13th annual Department of Energy Joint Genome Institute (JGI) “Genomics of Energy and Environment” meeting in San Francisco, CA. Three posters were presented: Chris Ward, “Towards an integrative understanding of chytrid parasitism and its drivers in mass algal culture,” Jeff Kimbrel, “Combining multiple functional annotation tools increases completeness of metabolic annotation,” and Xavier Mayali, “Nanoscale Stable Isotope Tracing to Investigate Interactions between Bacteria and Biofuel-producing Algae.” Mayali’s poster won the “Outside the Box Poster Award.” The LLNL team also presented two invited talks: “Exploring Microbial Ecology with Isotopes and Imaging” by Jennifer Pett-Ridge and “Exploring Metabolite Production from Tryptophan Precursors in Two Algal Associated Bacteria, Algoriphagus sp. ARW1R1 and Marinobacter sp. 19DW” by Ty Samo.

Visit the JGI website for more information.



LLNL’s Biofuels SFA is hiring!

We have a position for a postdoc to examine host-microbe metabolic interactions and exchange in microalgae and perennial grasses using metabolomics, stable isotope probing, and proteogenomic approaches. For more information, see position 103155 at https://jobs.llnl.gov. If you have questions, feel free to contact Michael Thelen at thelen1@llnl.gov.


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February 2018


New publication: High initial sputter rate found for Vaccinia virions using isotopic labeling, NanoSIMS, and AFM

Five correlated AFM and SIMS images of Vaccinia virions showing colocalization in the two instruments and general nature of virion surface erosion during sputtering.

Correlated AFM and SIMS images of Vaccinia virions showing colocalization in the two instruments and general nature of virion surface erosion during sputtering. The AFM image (A) is a contrast-enhanced height image that corresponds to the region in the SIMS images (B–E). The sequence of SIMS images detail the distribution of the 12C14N- species counts during each scan. SIMS image (B), (C), (D), and (E) correspond to scan number 1, 15, 25, and 39, respectively. The decrease in species counts as a function of scan number is a result of lateral erosion caused by sputtering. Scale bar: 500 nm.

New research supported by the LLNL Biofuels Scientific Focus Area (SFA) shows the promise and challenge of studying the role of viruses in microbial systems. Viruses are known to be ubiquitous and infect all forms of life, including microbes. As such, they are thought to have major roles in carbon and nutrient cycling, but to date, these rates have been unexplored in most systems. With this research, SFA researchers Peter Weber and Ben Stewart and colleagues explored the potential to use high-spatial resolution secondary ion mass spectrometry (SIMS) with a NanoSIMS 50 to characterize nutrient transfer from host to virus using isotopically labeled DNA as a tracer. Their work showed the expected transfer of isotopic label, which is promising for future application of SIMS to virus ecology, but they also found that at the start of SIMS analysis, these tiny structures eroded 100 times faster than previously expected. For the relatively large Vaccinia virus in this study, this was not a major problem, but for bacterial phage, which are as small as 20 nm in diameter, new methods may be necessary to ensure quality measurements of nutrient uptake and cycling.

[S. Gates, R.C. Condit, N. Moussatche, B.J. Stewart, A.J. Malkin, and P.K. Weber, High Initial Sputter Rate Found for Vaccinia Virions Using Isotopic Labeling, NanoSIMS, and AFM, Anal. Chem. 90 (3), 1613 (2018), doi: 10.1021/acs.analchem.7b02786]


SFA team members present at 2018 Ocean Sciences meeting

An ion image generated from the NanoSIMS.

An ion image generated from the NanoSIMS illustrates the high spatial resolution visualization of algal and bacterial biomass, as determined from 12C 14N ion masses. Using these images, exchanges of C and N between algae and bacteria as a function of their spatial interactions can be measured.

SFA members Xavier Mayali and Ty Samo attended the 2018 Ocean Sciences meeting in Portland, Oregon in February to present some of their work on algal-bacterial interactions using stable isotope probing and NanoSIMS. Ty presented a talk entitled “Stable Isotope Probing and NanoSIMS Reveals Effects of Physical Association on Mutualisms Between Individual Bacterial Cells and Two Species of Phytoplankton” in the session “Bridging Microbial, Stable Isotope, and Micronutrient Approaches to Marine Carbon and Nitrogen Recycling.” Xavier presented a talk entitled “Investigating C transfer between diatoms and phycosphere-associated bacteria with stable isotopes and NanoSIMS” in the session “Phytoplankton-Bacteria Interactions: From Microscales to Ocean Scales.”

Learn more about the conference on the 2018 Ocean Sciences Meeting site.



What Can Algae Do For Us?

In this video, SFA scientists help explain what algae are and what they can do for us.

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