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At least 289 records · Page 16

Divergent carbon use efficiency-growth rate tradeoff in popular biological growth models

Carbon use efficiency (CUE) is an important trait emerging from processes regulating biological growth. CUE can be computed either based on the growth of structural biomass or total biomass divided by substrate uptake rate. Nonequilibrium thermodynamics and observations suggest that, for an exponentially growing population of cells, structural biomass CUE should first increase, then peak, and finally decrease with specific growth rate; meanwhile, total biomass CUE increases asymptotically with specific growth rate. We compared predictions from six popular models that are often used for plant and microbial growth in existing ecosystem models. We found that, for an exponentially growing population of biological cells, (1) the source-driven Pirt and Compromise models predict that structural biomass CUE increase asymptotically with growth rate; (2) the apparent sink-driven modified Droop model predicts that structural biomass CUE decreases with growth rate; and (3) the sink-driven variable internal storage model and two dynamic energy budget models predict that structural biomass CUE first increases, then peaks, and finally decreases with growth rate. Moreover, the modified Droop model predicts that total biomass CUE is constant with growth rate, while all other five models predict that total biomass CUE increases with growth rate asymptotically. For non-exponential biological growth, we show that there is no static relationship between total biomass CUE or structural biomass CUE with respect to either growth rate or temperature. Therefore, we contend that biological growth models should explicitly represent interactions between substrate acquisition, substate transformation, and maintenance respiration to better capture observed CUE dynamics, and the sink-driven model should be preferred for general ecosystem biogeochemistry modeling.

Tang, Jinyun [Lawrence Berkeley National Laborator↗

Ecosystems and Networks Integrated with Genes and Molecular Assemblies (Final Scientific/Technical Report)

The ENIGMA science focus area (SFA) is a multi-disciplinary, multi-institutional research effort focused on addressing foundational knowledge gaps in environmental microbial communities by studying groundwater and sediment microbiomes in the shallow subsurface at the contaminated Oak Ridge Field Research Site (FRC). We seek to discover and characterize the reciprocal interactions between the microbial communities and the geochemical and geophysical parameters of the shallow subsurface within the contamination plume. The goal of the Fields lab is to help manage and conduct laboratory experiments informed by field observations using ecological and physiological approaches to study microbial populations in situ as well as ex situ (in the laboratory). In particular, we aim to study and characterize the impact of hydrological constraints on free-living and biofilm biomass and activity with increasing spatial and temporal resolution under static and flow conditions. We also aim to help characterize novel microbial groups that are present and active under relevant field conditions, including the development of molecular techniques for in situ detection as well as metabolic interactions that underlie pertinent physiology and ecology.

59 BASIC BIOLOGICAL SCIENCES↗

A Catabolic Powerhouse for Biorefineries: Characterization and Engineering Erwinia spp. Strain LJJL01 to Produce Bioproducts

Nonmodel microbial hosts can efficiently biotransform unconventional organic feedstocks into advanced bioproducts, leveraging their superior metabolic capabilities and resilience to process-relevant physicochemical conditions. In this study, we domesticated Erwinia spp. strain. LJJL01 (Er LJJL01) as a potent microbial chassis to advance the emerging biorefinery strategy for the valorization of lignin-rich biomass and plastic, thereby enabling the circular economy. The strain exhibits remarkable chemical tolerance and can biofunnel various substrates, including sugars, acids, polyols, and aromatics, in minimal salt media to produce native fine chemicals such as acetoin, 2,3-butanediol, and lactic acid. As a proof of concept, engineering this strain with advanced genetic tools enables the production of tailored high-value chemicals, such as cis,cis-muconate from plastic-derived terephthalate and polyhydroxybutyrate from lignocellulosic hydrolysate. We established Er LJJL01 as a potent microbial chassis for the green synthesis of bioproducts from unconventional organic feedstocks.

09 BIOMASS FUELS↗

In silico evaluation of a targeted metaproteomics strategy for broad screening of cellulolytic enzyme capacities in anaerobic microbiome bioreactors

Microbial-driven solubilization of lignocellulosic material is a natural mechanism that is exploited in anaerobic digesters (ADs) to produce biogas and other valuable bioproducts. Glycoside hydrolases (GHs) are the main enzymes that bacterial and archaeal populations use to break down complex polysaccharides in these reactors. Methodologies for rapidly screening the physical presence and types of GHs can provide information about their functional activities as well as the taxonomical diversity within AD systems but are largely unavailable. Targeted proteomic methods could potentially be used to provide snapshots of the GHs expressed by microbial consortia in ADs, giving valuable insights into the functional lignocellulolytic degradation diversity of a community. Such observations would be essential to evaluate the hydrolytic performance of a reactor or potential issues with it.

09 BIOMASS FUELS↗

Engineering Novosphingobium aromaticivorans to produce cis,cis -muconic acid from biomass aromatics

ABSTRACT The platform chemical cis,cis- muconic acid ( cc MA) provides facile access to a number of monomers used in the synthesis of commercial plastics. It is also a metabolic intermediate in the β-ketoadipic acid pathway of many bacteria and, therefore, a current target for microbial production from abundant renewable resources via metabolic engineering. This study investigates Novosphingobium aromaticivorans DSM12444 as a chassis for the production of cc MA from biomass aromatics. The N. aromaticivorans genome predicts that it encodes a previously uncharacterized protocatechuic acid (PCA) decarboxylase and a catechol 1,2-dioxygenase, which would be necessary for the conversion of aromatic metabolic intermediates to cc MA. This study confirmed the activity of these two enzymes in vitro and compared their activity to ones that have been previously characterized and used in cc MA production. From these results, we generated one strain that is completely derived from native genes and a second that contains genes previously used in microbial engineering synthesis of this compound. Both of these strains exhibited stoichiometric production of cc MA from PCA and produced greater than 100% yield of cc MA from the aromatic monomers that were identified in liquor derived from alkaline pretreated biomass. Our results show that a strain completely derived from native genes and one containing homologs from other hosts are both capable of stoichiometric production of cc MA from biomass aromatics. Overall, this work combines previously unknown aspects of aromatic metabolism in N. aromaticivorans and the genetic tractability of this organism to generate strains that produce cc MA from deconstructed biomass. IMPORTANCE The production of commodity chemicals from renewable resources is an important goal toward increasing the environmental and economic sustainability of industrial processes. The aromatics in plant biomass are an underutilized and abundant renewable resource for the production of valuable chemicals. However, due to the chemical composition of plant biomass, many deconstruction methods generate a heterogeneous mixture of aromatics, thus making it difficult to extract valuable chemicals using current methods. Therefore, recent efforts have focused on harnessing the pathways of microorganisms to convert a diverse set of aromatics into a single product. Novosphingobium aromaticivorans DSM12444 has the native ability to metabolize a wide range of aromatics and, thus, is a potential chassis for conversion of these abundant compounds to commodity chemicals. This study reports on new features of N. aromaticivorans that can be used to produce the commodity chemical cis,cis -muconic acid from renewable and abundant biomass aromatics.

09 BIOMASS FUELS↗

Resilient Crop Production for the Bioeconomy: Frontier Science for the Bioeconomy Workshop Series

A bioeconomy based on energy, chemicals, and bioproducts derived from nonfood crops promotes U.S. prosperity, energy independence, and national security. Crop productivity (i.e., yield) affects profitability for farmers, whereas biomass and seed oil quality affects profitability for biorefineries through the conversion efficiency of feedstock crops into energy, chemicals, and bioproducts. Scientific breakthroughs in plant genetics and genomics, ecological processes, bioprocessing, microbial engineering and design, and conversion technologies have improved the potential market viability of products derived from nonfood crops specifically cultivated as biomass and seed oil feedstocks. Improving understanding of a plant’s complex responses to withstand or recover from stress is crucial to maintaining feedstock yield and quality, particularly in challenging production zones. Stress may be related to biotic or abiotic (i.e., nonliving) environmental conditions that negatively affect plant growth, development, and productivity. Research and development to enhance the environmental resilience of feedstock crops will enable improved production by minimizing losses during stress and improving production on currently economically nonviable marginal lands.

09 BIOMASS FUELS↗

Waste-to-Energy Pipeline through Consolidated Fermentation–Microbial Fuel Cell (MFC) System

The rise in population, urbanization, and industrial developments have led to a substantial increase in waste generation and energy demand, posing significant challenges for waste management as well as energy conservation and production. Bioenergy conversions have been merged as advanced, sustainable, and integrated solutions for these issues, encompassing energy generation and waste upcycling of different types of organic waste. Municipal solid waste (MSW) and agricultural residues (AR) are two main resources for bioenergy conversions. Bioenergy production involves feedstock deconstruction and the conversion of platform chemicals to energy products. This review provides a detailed overview of waste sources, biofuel, and bioelectricity production from fermentation and microbial fuel cell (MFC) technology, and their economic and environmental perspectives. Fermentation plays a critical role in liquid biofuel production, while MFCs demonstrate promising potential for simultaneous production of electricity and hydrogen. Fermentation and MFCs hold a significant potential to be integrated into a single pipeline, enabling the conversion of organic matter, including a variety of waste material and effluent, into diverse forms of bioenergy via microbial cultures under mild conditions. Furthermore, MFCs are deemed a promising technology for pollutant remediation, reducing COD levels while producing bioenergy. Importantly, the consolidated fermentation–MFC system is projected to produce approximately 7.17 trillion L of bioethanol and 6.12 × 10 4 MW/m 2 of bioelectricity from MSW and AR annually, contributing over USD 465 billion to the global energy market. Such an integrated system has the potential to initiate a circular economy, foster waste reduction, and improve waste management practices. This advancement could play a crucial role in promoting sustainability across the environmental and energy sectors.

09 BIOMASS FUELS↗

Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two–stage fine chemical production with Kluyveromyces marxianus

Development of the bioeconomy is driven by our ability to access the energy-rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value-added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two-stage bioprocessing system pairing the anaerobic fungus Piromyces indianae with the yeast Kluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose. P. indianae efficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, which K. marxianus consumed while producing 2.4 g/L ethyl acetate. An engineered strain of K. marxianus was also able to produce 550 mg/L 2-phenylethanol and 150 mg/L isoamyl alcohol from P. indianae hydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. Furthermore, this work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.

59 BASIC BIOLOGICAL SCIENCES↗

Mapping microhabitats of lignocellulose decomposition by a microbial consortium

The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Assessing microbial systems and process configurations for improved ethanol production from sugary stovers by integrating soluble sugars and holocellulose

Here, this study evaluated microbial systems and technological approaches to configure the whole slurry co-fermentation process for ethanol biosynthesis from a novel stover system rich in sugars. Two approaches, namely separate and simultaneous hydrolysis and co-fermentation (SHCF and SSCF, respectively), were investigated using Escherichia coli monoculture and E. coli-yeast coculture. The SSCF with E. coli monoculture produced 32.75 g/L ethanol, representing only 44.87% yield, which left 65.13 g/L of total sugars unconverted and exhibited limited xylose consumption. Subsequently, a coculture-based SHCF significantly enhanced sugar consumption, leading to increase in ethanol yield and concentration to 66.94% and 48.86 g/L, respectively. Nevertheless, xylose utilization remained minimal due to the preference for glucose and the inhibitory effects of certain compounds. Thereafter, modification of the medium composition by supplementing betaine and sodium metabisulfite improved ethanol production to 53.18 g/L by reducing the toxic effects of inhibitors. Finally, a dual-phase SSCF (DP-SSCF) was explored by allowing the consumption of sugars from the pretreated slurry in the first phase, followed by concurrent cellulose hydrolysis and utilization of the resulting glucose in the second phase. This strategy increased ethanol titer to 63.14 g/L, with 84.3% yield and 0.88 g/L/h productivity.

09 BIOMASS FUELS↗

Direct root contact among neighboring plants influences activity of soil extracellular enzymes

Composition and diversity of vegetation systems can influence soil microbial activity and extracellular enzyme (EE) dynamics, which are crucial for soil carbon (C) accrual and nutrient cycling. Yet, the impact of plant interactions and competition on EE activities remains a notable knowledge gap. This study examines how direct root contact and neighboring plant identity affect the activity and spatial distribution of four key soil EEs: β-glucosidase (BGlu), chitinase, acid phosphatase (AcidP), and alkaline phosphatase (AlkP). Using three-compartment rhizoboxes with switchgrass (Panicum virgatum L.) grown alongside bush clover (Lespedeza capitata Michx.), and black-eyed Susan (Rudbeckia hirta L.), we assessed enzyme activities using zymography under conditions that either allowed or restricted direct root contact by root barriers. Results show that root proliferation and species interactions significantly influenced EE activity. While BGlu and AcidP activities were strongly correlated with root biomass, AlkP activity was consistently higher in the absence of root barriers, indicating a pronounced microbial response to plant interactions via direct/close root contacts. Additionally, soil phosphorus availability modulated enzyme activity, with higher phosphatase activities in low-P soils. Furthermore, these findings highlight the importance of root-root interactions and plant species composition in shaping soil biochemical processes.

enyzme activity↗

Steam explosion pretreatment coupling high-temperature short-time sterilization facilitating cellulose degradation and sporulation-regulatory gene expression in high-solid fermentation

Steam explosion coupling high-temperature short-time sterilization (SE-HTST) was exploited to modify cellulosic biomass medium properties and promote high-solid fermentation (HSF). Biomass characterization analysis showed that SE-HTST enlarged microstructural pores and cavities in solid media, providing more effective space for microbial growth. Meanwhile, SE-HTST helped to release glucose from the cellulose with 35.8 ± 4.5, 20.0 ± 2.3, and 12.3 ± 5.7 mg glucose/g dry medium at 24, 48, and 72 h of fermentation, which were 3.1, 2.3, and 1.5 times higher than that in medium from conventional thermal sterilization (CTS), respectively. SE-HTST increased the viable cell and spore number of Bacillus subtilis by 1.8 and 1.6 times at 72 h of fermentation compared to CTS. Moreover, the expressions of master transcriptional gene spo0A and the early sigma factors of sigF and sigE genes gradually increased in the SE-HTST medium, showing enhanced sporulation in HSF. Therefore, SE-HTST is an effective strategy for facilitating cellulose degradation, improving glucose nutrients in biomass medium, and promoting sporulation-regulatory gene expression during high-solid fermentation, which enhances the production of microbial ecological agents using B. subtilis significantly.

59 BASIC BIOLOGICAL SCIENCES↗

Risk Assessment of Industrial Microbes Using a Terrestrial Mesocosm Platform

Abstract Industrial microbes and bio-derived products have emerged as an integral component of the bioeconomy, with an array of agricultural, bioenergy, and biomedical applications. However, the rapid development of microbial biotechnology raises concerns related to environmental escape of laboratory microbes, detection and tracking thereof, and resultant impact upon native ecosystems. Indeed, though wild-type and genetically modified microbes are actively deployed in industrial bioprocesses, an understanding of microbial interactivity and impact upon the environment is severely lacking. In particular, the persistence and sustained ecosystem impact of industrial microbes following laboratory release or unintentional laboratory escape remains largely unexplored. Herein, we investigate the applicability of soil-sorghum mesocosms for the ecological risk assessment of the industrial microbe, Saccharomyces cerevisiae . We developed and applied a suite of diagnostic and bioinformatic analyses, including digital droplet PCR, microscopy, and phylogenomic analyses to assess the impacts of a terrestrial ecosystem perturbation event over a 30-day time course. The platform enables reproducible, high-sensitivity tracking of S. cerevisiae in a complex soil microbiome and analysis of the impact upon abiotic soil characteristics and soil microbiome population dynamics and diversity. The resultant data indicate that even though S. cerevisiae is relatively short-lived in the soil, a single perturbation event can have sustained impact upon mesocosm soil composition and underlying microbial populations in our system, underscoring the necessity for more comprehensive risk assessment and development of mitigation and biocontainment strategies in industrial bioprocesses.

09 BIOMASS FUELS↗

Practical and thermodynamic constraints on electromicrobially accelerated CO 2 mineralization

By the end of the century, tens of gigatonnes of CO 2 will need to be removed from the atmosphere every year to maintain global temperatures. Natural weathering of ultramafic rocks and subsequent mineralization reactions can convert CO 2 into ultra-stable carbonates. Although this will draw down all excess CO 2 , it will take thousands of years. CO 2 mineralization could be accelerated by weathering ultramafic rocks with biodegradable lixiviants. We show that if these lixiviants come from cellulosic biomass, this demand could monopolize the world’s biomass supply. We demonstrate that electromicrobial production technologies (EMP) that combine renewable electricity and microbial metabolism could produce lixiviants for as little as $\$200$ to $\$400$ per tonne at solar electricity prices achievable within the decade. We demonstrate that EMP could make enough lixiviants to sequester a tonne of CO 2 for less than $\$100$. This work highlights the potential of this approach and the need for extensive R&D.

59 BASIC BIOLOGICAL SCIENCES↗

Trophic interactions shape the spatial organization of medium-chain carboxylic acid producing granular biofilm communities

Abstract Granular biofilms producing medium-chain carboxylic acids (MCCA) from carbohydrate-rich industrial feedstocks harbor highly streamlined communities converting sugars to MCCA either directly or via lactic acid as intermediate. We investigated the spatial organization and growth activity patterns of MCCA producing granular biofilms grown on an industrial side stream to test (i) whether key functional guilds (lactic acid producing Olsenella and MCCA producing Oscillospiraceae) stratified in the biofilm based on substrate usage, and (ii) whether spatial patterns of growth activity shaped the unique, lenticular morphology of these biofilms. First, three novel isolates (one Olsenella and two Oscillospiraceae species) representing over half of the granular biofilm community were obtained and used to develop FISH probes, revealing that key functional guilds were not stratified. Instead, the outer 150–500 µm of the granular biofilm consisted of a well-mixed community of Olsenella and Oscillospiraceae, while deeper layers were made up of other bacteria with lower activities. Second, nanoSIMS analysis of 15N incorporation in biofilms grown in normal and lactic acid amended conditions suggested Oscillospiraceae switched from sugars to lactic acid as substrate. This suggests competitive-cooperative interactions may govern the spatial organization of these biofilms, and suggests that optimizing biofilm size may be a suitable process engineering strategy. Third, growth activities were similar in the polar and equatorial biofilm peripheries, leaving the mechanism behind the lenticular biofilm morphology unexplained. Physical processes (e.g., shear hydrodynamics, biofilm life cycles) may have contributed to lenticular biofilm development. Together, this study develops an ecological framework of MCCA-producing granular biofilms that informs bioprocess development.

09 BIOMASS FUELS↗

Integrating catalytic fractionation and microbial funneling to produce 2-pyrone-4,6-dicarboxylic acid and ethanol

Replacing biorefinery designs that use stepwise fractionation, depolymerization, and conversion processes with designed tandem process steps can greatly reduce a biorefinery's operating costs and environmental impact. Reductive Catalytic Fractionation (RCF) is a highly efficient lignin-first approach that combines biomass fractionation and lignin depolymerization to generate a hydrogenolysis oil and pulp. The oil is composed of a complex mixture of phenolic monomers, dimers, and oligomers. Intergrating this chemical deconstruction process with microbial funneling of phenolics can simplify the product mixture and make high-value products. We applied RCF to poplar biomass in a biomass-to-bioproduct processing chain in which the phenolics were funneled to 2-pyrone-4,6-dicarboxylic acid (PDC) by an engineered strain of Novosphingobium aromaticivorans DSM12444. The pulp was enzymatically digested and the glucose and xylose was funneled to ethanol by an engineered strain of Saccharomyces cerevisiae GLBRCY945. By combining biomass fractionation and lignin depolymerization we removed a costly processing step that directly translated into a 29% reduction in the minimum selling price of PDC. This work combines experimentation with process modeling of an integrated biorefinery design to show how utilizing tandem process steps can significantly reduce operating expenses and environmental impact of upgrading lignocellulosic biomass to a portfolio of high value products.

Sener, Canan [Great Lakes Bioenergy Research Cente↗

Development and implementation of an in situ high-resolution isotopic microscope for measuring metabolic interactions in soil mesocosms (Final Report)

Many of our planet’s ecosystems rely on the activities of soil microbial communities. These microbes have significant and wide-ranging effects: they metabolize carbon and other nutrients, interact with plants and fungi, and perform other processes important to soil health. However, our ability to directly observe the enzymatic and metabolic activities of microbes within soil is currently limited, not only by the complexity of soil microbial communities themselves, but also by the lack of experimental tools to study them and their molecular interactions in situ. These challenges hinder our understanding of the life-sustaining processes of biomass decomposition and the manner in which it contributes to the movement of freed carbon within soil ecosystems. Here we aimed to develop a novel ultrahigh-resolution isotopic microscope that combines complementary imaging modalities to gain insights into metabolic cycling in soil. This final report covers both the portion of this work that was initially completed at the University of North Carolina at Chapel Hill from 2018 to 2019 (as award DE-SC0019012) and then from 2020 to 2022 (2023 in NCE) at the University of Massachusetts Chan Medical School. This goal was to build an integrated platform consisting of fluorescence microscopy, Raman microspectroscopy, and nanospray desorption electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoDESI-FTICR-MS) to directly investigate microbial activities and molecular transformations occurring in soil by exploiting the use of both fluorescent labels and stable isotope probing. Specifically, our instrument was conceived as integrating: (A) fluorescence detection to localize soil microbes, identify bacteria taking up polysaccharides, and monitor gene expression of enzymes involved in decomposition; (B) Raman microspectroscopy to determine which microbes incorporate decomposition products into their biomass, as well as which decomposers are metabolically active; and (C) nanoDESI-FTICR-MS imaging to spatially probe, in real-time, the metabolites in the surrounding area, which we expect will reveal the distributions of the products of the enzymatic breakdown of polysaccharides, as well as specialized metabolites acting as cell-cell signals between decomposers. To enable these measurements, we exploited fluorescence-based probes to map the microbes that are enzymatically active (per A) and utilized stable isotope-labeled substrates to visualize both the microbial and molecular fate of decomposed biomass (B and C, respectively). To accomplish our goal, we combined the expertise of a multidisciplinary group of scientists to pursue the construction of this microscope and to investigate scientific questions that would be facilitated by such a capability. We aimed to employ this spatially informative, high-resolution isotopic microscope to visualize the critical steps of biomass degradation and the molecular fate of other environmentally relevant substrates within soil mesocosms. Accordingly, this technology will enhance our understanding of the microbial and metabolic interactions occurring within soil communities that are relevant to carbon degradation and other soil processes. The instrument has been developed and housed at EMSL, where it will be available to the entire EMSL User Base, enabling a variety of related DOE-relevant systems to be interrogated in the future by diverse scientific research groups.

59 BASIC BIOLOGICAL SCIENCES↗