Bottleneck/Challenge: The high complexity and multitude of soil microbes make it difficult to identify soil microbial community function using currently available -omics techniques.

Potential Solution: Develop high-throughput proteomics techniques to better understand biological functions in soil matrix.

Potential Solution: Improve soil metabolomics reference databases and metabolomics techniques to better understand biogeochemical cycling in soil.

Potential Solution: Integrate multiple -omics datasets into a single database.

Bottleneck/Challenge:  Paucity of models for how soil microbiota regulate soil carbon capacity across different spatial (microscopic, mesoscopic, and macroscopic) and temporal scales.

Potential Solution:  Develop tools to enable the measurement of biochemical reactions (microscale) in the field (macroscale).

Potential Solution:  Consolidate datasets from lab- and field-based studies to create an integrated soil microbiome database.

Potential Solution:  Develop computational models (informed by lab and field studies) to bridge the knowledge gaps between different spatial and temporal scales.

Bottleneck/Challenge: Limited understanding of how microbes deposit carbon as minerals and sediments in soil.

Potential Solution: Develop metabolite labeling and tracing tools that can be used to track carbon movement in soil microbiome first in laboratory conditions and later in situ.⁸Watts-Williams, S. J. (2022). Track and trace: How soil labelling techniques have revealed the secrets of resource transport in the arbuscular mycorrhizal symbiosis. Mycorrhiza, 32(3), 257–267. View Publication.

Bottleneck/Challenge: Current poor understanding of the chemical and ecological factors that govern the residence time of specific molecules in soils (e.g., betaine).

Potential Solution: Highly controlled ecosystem studies coupled to high resolution mass spectrometry to determine the factors that affect the turnover of specific molecules in soils.

Bottleneck/Challenge: Avoiding undesired phenotypes associated with engineering metabolic flux.⁹Mahmood, K., Zeisler-Diehl, V. V., Schreiber, L., Bi, Y.-M., Rothstein, S. J., & Ranathunge, K. (2019). Overexpression of ANAC046 Promotes Suberin Biosynthesis in Roots of Arabidopsis thaliana. International Journal of Molecular Sciences, 20(24), 6117. View Publication.^{, 10}Baxter, I., Hosmani, P. S., Rus, A., Lahner, B., Borevitz, J. O., Muthukumar, B., Mickelbart, M. V., Schreiber, L., Franke, R. B., & Salt, D. E. (2009). Root Suberin Forms an Extracellular Barrier That Affects Water Relations and Mineral Nutrition in Arabidopsis. PLOS Genetics, 5(5), e1000492. View Publication.

Potential Solution:  Genetic determinants for some carbon-storing compounds are well characterized (see Harman-Ware et al., 2021¹¹Harman-Ware, A. E., Sparks, S., Addison, B., & Kalluri, U. C. (2021). Importance of suberin biopolymer in plant function, contributions to soil organic carbon and in the production of bio-derived energy and materials. Biotechnology for Biofuels, 14(1), 75. View Publication.); apply this understanding to systems/organismal engineering to enable control of compound synthesis, transport, and storage.

Bottleneck/Challenge:  The synthesis of carbon-storing compounds can vary by cultivar in response to environmental conditions.

Potential Solution:  Undertake large field trials using genomic, transcriptomic, proteomic, and metabolomic analyses to understand the contributions of genetics and the environment to the synthesis of key carbon-storing compounds, such as suberin, in non-model and crop plants.¹²Harman-Ware, A. E., Sparks, S., Addison, B., & Kalluri, U. C. (2021). Importance of suberin biopolymer in plant function, contributions to soil organic carbon and in the production of bio-derived energy and materials. Biotechnology for Biofuels, 14(1), 75. View Publication.

Potential Solution:  Understand and engineer synthesis and transport mechanisms for carbon-storing compounds, such as suberin, that are synthesized above and below ground for consistent root accumulation across environmental conditions and relevant plant cultivars.

Potential Solution:  Engineer suberin to increase carrying capacity and retain carbon for longer time periods.

Bottleneck/Challenge:  Relationships between plants and microbes vary under differing environmental conditions, potentially to the plant’s benefit (see von Rein et al., 2016¹³von Rein, I., Gessler, A., Premke, K., Keitel, C., Ulrich, A., & Kayler, Z. E. (2016). Forest understory plant and soil microbial response to an experimentally induced drought and heat-pulse event: The importance of maintaining the continuum. Global Change Biology, 22(8), 2861–2874. View Publication.; Wipf et al., 2021¹⁴Wipf, H. M.-L., Bùi, T.-N., & Coleman-Derr, D. (2021). Distinguishing Between the Impacts of Heat and Drought Stress on the Root Microbiome of Sorghum bicolor. Phytobiomes Journal, 5(2), 166–176. View Publication.); preferencing the recruitment of target microbes under stressful environmental conditions may disrupt interactions that support plant health.

Potential Solution:  Engineer microbiomes that are responsive to changing conditions.

Bottleneck/Challenge: Plant metabolites may need to be converted to alternative forms or compounds by microbiome community members in order to recruit microbes that increase carbon capacity, which would be challenging to track and elucidate.

Potential Solution:  Enhance capabilities for tracing metabolite transfer from plants through the microbiome, working toward greater resolution (e.g., Family or Genus) of involved community members.

Potential Solution: Harness microbial communities and knowledge of specific molecules with longer residence times under specific environmental and ecological conditions.

Potential Solution: Maximize microbial conversion of exudate to biomass (and subsequently, microbial necromass) by designing microbial communities that use all exudate components.

Bottleneck/Challenge:  Unstable soil aggregates (i.e., due to tilling) release captured carbon back into the atmosphere.

Potential Solution:  Engineer soil microbes to produce biofilms to promote the formation of soil aggregates.

Potential Solution:  Engineer biomaterials to stabilize soil micro-aggregates.

Bottleneck/Challenge: Fungal hyphal networks (e.g,. arbuscular mycorrhizal fungi) have been shown to enhance mineral weathering in soil, but the most well-studied of such fungi – mycorrhizal fungi –need host plants to survive.

Potential Solution:  Engineer arbuscular mycorrhizal fungi to survive independent of a host plant.

Potential Solution:  Develop means to seed soil with fungi capable of mineral weathering (e.g., saprotrophic fungi) and surviving without host plants.

Bottleneck/Challenge:  The exometabolome is challenging to characterize because of extensive cross-feeding/uptake by other community members.

Potential Solution:  Develop improved tools to quantify the exchange of metabolites within complex microbiomes.¹⁵Douglas, A. E. (2020). The microbial exometabolome: Ecological resource and architect of microbial communities. Philosophical Transactions of the Royal Society B: Biological Sciences, 375(1798), 20190250. View Publication.

Bottleneck/Challenge: Uncharacterized permafrost microbiome.

Potential Solution: Use multi-omics tools and machine learning to build comprehensive datasets of permafrost microbial communities.

Bottleneck/Challenge: Relevant soil microbiome constituents are under-characterized and we lack understanding of which species might be best for engineering approaches.

Potential Solution: Expand capabilities for culturing recalcitrant microbes.

Potential Solution: Develop techniques to characterize unculturable microbes in situ.

Bottleneck/Challenge: More understanding is needed on the durability of sequestered carbon and how biocrust interacts with other parts of the carbon cycle.

Potential Solution:  Identify biochemical factors that increase carbon sequestration in biocrusts.

Potential Solution:  Measure carbon sequestration capacity in artificial biocrust over temporal and spatial scales, and under different environmental conditions (e.g., temperature, precipitation, and nutrient levels).

Bottleneck/Challenge:  It is difficult to isolate fast-growing and suitable cyanobacteria for inoculating biocrust.

Potential Solution:  Identify, cultivate, and engineer filamentous cyanobacteria from a variety of dryland regions for desired growth rate and robustness under requisite environmental conditions (e.g., high summer heat).

Potential Solution: Develop and engineer consortia of cyanobacteria (different species) to more successfully inoculate artificial or native biocrusts.

Bottleneck/Challenge: Processes for inoculating engineered cyanobacteria into natural biocrusts are underdeveloped.

Bottleneck/Challenge: Biocrusts can be incredibly complex depending on the organisms/species and abiotic components involved; the interaction with native ecosystems would need to be structured and resolved.

Potential Solution: Characterize and engineer symbiosis between plants and artificially enhanced biocrusts to increase soil stability.

Bottleneck/Challenge: Mechanisms of microbial ice nucleation are not well understood.

Potential Solution: Use advanced -omics techniques to identify links between microbes/microbial communities and their ability to nucleate ice.

Bottleneck/Challenge: A lack of genetic editing tools to engineer ice nucleating biological systems.

Potential Solution: Develop genetic tools (e.g., transformation methods, genetic parts) for engineering cryophilic microbial chassis.

Potential Solution: Identify or design ice nucleating proteins with high efficiency and robustness of ice nucleation.

Bottleneck/Challenge: Poor understanding of environmental factors that inhibit or enhance microbial ice nucleation.

Potential Solution:  Utilize machine learning and artificial intelligence to design models for microbial ice nucleation based on data from persistent, native microbial communities.

Bottleneck/Challenge:  Poor understanding how ice nucleating microbes interact with the broader ecosystem (for example, the role of ice nucleating bacteria in arctic marine environments).

Potential Solution:  Develop and test strategies for biocontainment of engineered microbes in the polar/alpine environment.

Bottleneck/Challenge: Limited understanding of microbial carbon-cycling processes in anaerobic and/or high-salinity soils.

Potential Solution: Extend metagenomics, proteomics, and metabolomics tools for studying soils to wetland soil environments.

Bottleneck/Challenge: Limited understanding of how ecological stressors impact marine microbes in coastal ecosystems on a genetic and metabolic level.

Potential Solution: Engineer field-deployable biosensors for local chemistry (e.g., salinity) and pollutants (e.g., agricultural fertilizer runoff) that specifically impacts wetlands, salt marshes, and other coastal ecosystems.

Bottleneck/Challenge: Limited gene editing tools for engineering macroalgae (e.g., Saccharina and Gracilaria).

Potential Solution: Develop and improve direct bacterial transformation (such as in ways similar to agrobacterium transformation of plants).¹⁶Oertel, W., Wichard, T., & Weissgerber, A. (2015). Transformation of Ulva mutabilis (Chlorophyta) by vector plasmids integrating into the genome. Journal of Phycology, 51(5), 963–979. View Publication.

Potential Solution: Gain better control of gametophyte hybridization and development of CRISPR-Cas systems.¹⁷Wang, X., Yao, J., Zhang, J., & Duan, D. (2020a). Status of genetic studies and breeding of Saccharina japonica in China. Journal of Oceanology and Limnology, 38(4), 1064–1079. View Publication.

Bottleneck/Challenge: Macroalgae grown in offshore environments compete for nutrients with carbon-fixing phytoplankton.

Potential Solution: Deploy carbon-capturing microbes that produce nutrients necessary for macroalgae growth in coastal algal farms.

Bottleneck/Challenge: Paucity of information about (but ever-increasing volume of) refractory organic compounds and chemical structures.

Potential Solution: Develop further understanding of and engineer microbes that enhance processes involved in the biological carbon pump that leads to long-term sequestration of carbon from the surface ocean to the deep ocean interiors.

Potential Solution: Identify how conversion of short-lived organic pools to recalcitrant carbon impacts microbial-driven nutrient cycles.

Potential Solution: Develop further understanding of how bacteria, viruses, plankton, and other microbes interact to facilitate long-term carbon sequestration.