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At least 307 records · Page 17

The microbicidal potential of visible blue light in clinical medicine and public health

Visible blue light of wavelengths in the 400–470 nm range has been observed to have microbicidal properties. A widely accepted hypothesis for the mechanism of microbial inactivation by visible blue light is that the light causes photoexcitation of either endogenous (present within the microbe) or, exogenous (present in the biological medium surrounding the microbe) photosensitizers such as porphyrins and flavins, which leads to the release of reactive oxygen species that subsequently manifests microbicidal activity. Some of the factors that have been observed to be associated with enhanced microbicidal action include increased duration of exposure, and either pre- or co-treatment with quinine hydrochloride. In case of bacteria, repetitive exposure to the blue light shows no significant evidence of resistance development. Additionally, visible blue light has exhibited the ability to inactivate fungal and viral pathogens and, multidrug-resistant bacteria as well as bacterial biofilms. Visible blue light has demonstrated efficacy in eliminating foodborne pathogens found on food surfaces and exposed surfaces in the food processing environment as well as in the decontamination of surfaces in the clinical environment to minimize the spread of nosocomial infections. We conclude from reviewing existing literature on the application of the blue light in clinical medicine and public health settings that this microbicidal light is emerging as a safer alternative to conventional ultraviolet light-based technologies in multiple settings. However, further comprehensive studies and thorough understanding of the mechanism of microbicidal action of this light in different scenarios is warranted to determine its place in human health and disease.

60 APPLIED LIFE SCIENCES↗

Microbial Diversity Associated with the Pollen Stores of Captive-Bred Bumble Bee Colonies

The pollen stores of bumble bees host diverse microbiota that influence overall colony fitness. Yet, the taxonomic identity of these symbiotic microbes is relatively unknown. In this descriptive study, we characterized the microbial community of pollen provisions within captive-bred bumble bee hives obtained from two commercial suppliers located in North America. Findings from 16S rRNA and ITS gene-based analyses revealed that pollen provisions from the captive-bred hives shared several microbial taxa that have been previously detected among wild populations. While diverse microbes across phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Ascomycota were detected in all commercial hives, significant differences were detected at finer-scale taxonomic resolution based on the supplier source. The causative agent of chalkbrood disease in honey bees, Ascosphaera apis, was detected in all hives obtained from one supplier source, although none of the hives showed symptoms of infection. The shared core microbiota across both commercial supplier sources consisted of two ubiquitous bee-associated groups, Lactobacillus and Wickerhamiella/Starmerella clade yeasts that potentially contribute to the beneficial function of the microbiome of bumble bee pollen provisions.

16S rRNA gene↗

The Promises, Challenges, and Opportunities of Omics for Studying the Plant Holobiont

Microorganisms are critical drivers of biological processes that contribute significantly to plant sustainability and productivity. In recent years, emerging research on plant holobiont theory and microbial invasion ecology has radically transformed how we study plant–microbe interactions. Over the last few years, we have witnessed an accelerating pace of advancements and breadth of questions answered using omic technologies. Herein, we discuss how current state-of-the-art genomics, transcriptomics, proteomics, and metabolomics techniques reliably transcend the task of studying plant–microbe interactions while acknowledging existing limitations impeding our understanding of plant holobionts.

59 BASIC BIOLOGICAL SCIENCES↗

Optimization of Molecular Methods for Detecting Duckweed-Associated Bacteria

The bacterial colonization dynamics of plants can differ between phylogenetically similar bacterial strains and in the context of complex bacterial communities. Quantitative methods that can resolve closely related bacteria within complex communities can lead to a better understanding of plant–microbe interactions. However, current methods often lack the specificity to differentiate phylogenetically similar bacterial strains. In this study, we describe molecular strategies to study duckweed–associated bacteria. We first systematically optimized a bead-beating protocol to co-isolate nucleic acids simultaneously from duckweed and bacteria. We then developed a generic fingerprinting assay to detect bacteria present in duckweed samples. To detect specific duckweed–bacterium associations, we developed a genomics-based computational pipeline to generate bacterial strain-specific primers. These strain-specific primers differentiated bacterial strains from the same genus and enabled the detection of specific duckweed–bacterium associations present in a community context. Moreover, we used these strain-specific primers to quantify the bacterial colonization of duckweed by normalization to a plant reference gene and revealed differences in colonization levels between strains from the same genus. Lastly, confocal microscopy of inoculated duckweed further supported our PCR results and showed bacterial colonization of the duckweed root–frond interface and root interior. The molecular methods introduced in this work should enable the tracking and quantification of specific plant-microbe associations within plant-microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗

Whole‐Cell MALDI‐ToF MS Coupled with Untargeted Metabolomics Facilitates Investigations of Microbial Chemical Interactions

Abstract The emergence of drug‐resistant pathogens necessitates the development of new countermeasures. In this regard, the introduction of probiotics to directly attack or competitively exclude pathogens presents a useful strategy. Application of this approach requires an understanding of how a probiotic and its target pathogen interact. A key means of probiotic‐pathogen interaction involves the production of small molecules called natural products (NPs). Here, we report the use of whole‐cell matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐ToF) mass spectrometry to characterize NP production by candidate probiotics (mouse airway microbiome isolates) when co‐cultured with the respiratory pathogen Burkholderia . We found that a Bacillus velezensis strain inhibits growth of and elicits NP production by Burkholderia thailandensis . Dereplication of known NPs detected in the metabolome of this B. velezensis strain suggests that a previously unannotated bioactive compound is involved. Thus, we present the use of whole‐cell MALDI as a broadly applicable method for screening the NP composition of microbial co‐cultures; this can be combined with other ‐omics methods to characterize probiotic‐pathogen and other microbe‐microbe interactions.

Aiosa, Nicole↗

Activity‐Based Protein Profiling – Enabling Phenotyping of Host‐Associated and Environmental Microbiomes

Abstract Host‐associated and environmental microbiomes play central roles in human health, biogeochemical cycling, response to ecosystem change, and agriculture. Scientific approaches that can reveal the functional activities that contribute to observed phenotypes are needed in microbiome research. Broad characterization of the functional activity of microbes within microbiomes is currently hampered by approaches that rely on inference of function from metagenomes or indirect measurements. Activity‐based protein profiling is uniquely positioned to overcome these challenges and reveal the protein‐level mechanisms associated with microbiome phenotypes. In this review we describe the progress made to date using ABPP in gut microbiome, plant‐microbe interaction, and soil microbiome research, and suggest how ABPP data can be coupled with advanced computational methods to enable phenotype prediction and bioengineering of microbiomes for applied purposes.

59 BASIC BIOLOGICAL SCIENCES↗

An ecological framework for microbial metabolites in the ocean ecosystem

The ocean microbe‐metabolite network involves thousands of individual metabolites that encompass a breadth of chemical diversity and biological functions. These microbial metabolites mediate biogeochemical cycles, facilitate ecological relationships, and impact ecosystem health. While analytical advancements have begun to illuminate such roles, a challenge in navigating the deluge of marine metabolomics information is to identify a subset of metabolites that have the greatest ecosystem impact. Here, we present an ecological framework to distill knowledge of fundamental metabolites that underpin marine ecosystems. We borrow terms from macroecology that describe important species, namely “dominant,” “keystone,” and “indicator” species, and apply these designations to metabolites within the ocean microbial metabolome. These selected metabolites may shape marine community structure, function, and health and provide focal points for enhanced study of microbe‐metabolite networks. Applying ecological concepts to marine metabolites provides a path to leverage metabolomics data to better describe and predict marine microbial ecosystems.

microbial metabolites↗

Substrate, temperature, and geographical patterns among nearly 2000 natural yeast isolates

Yeasts have broad importance as industrially and clinically relevant microbes and as powerful models for fundamental research, but we are only beginning to understand the roles yeasts play in natural ecosystems. Yeast ecology is often more difficult to study compared to other, more abundant microbes, but growing collections of natural yeast isolates are beginning to shed light on fundamental ecological questions. In this work, we used environmental sampling and isolation to assemble a dataset of 1962 isolates collected from throughout the contiguous United States of America (USA) and Alaska, which were then used to uncover geographic patterns, along with substrate and temperature associations among yeast taxa. We found some taxa, including the common yeasts Torulaspora delbrueckii and Saccharomyces paradoxus, to be repeatedly isolated from multiple sampled regions of the USA, and we classify these as broadly distributed cosmopolitan yeasts. A number of yeast taxon-substrate associations were identified, some of which were novel and some of which support previously reported associations. Further, we found a strong effect of isolation temperature on the phyla of yeasts recovered, as well as for many species. We speculate that substrate and isolation temperature associations reflect the ecological diversity of and niche partitioning by yeast taxa.

59 BASIC BIOLOGICAL SCIENCES↗

The Epidermal Microbiome Within an Aggregation of Leopard Sharks ( Triakis semifasciata ) Has Taxonomic Flexibility with Gene Functional Stability Across Three Time-points

The epidermis of Chondrichthyan fishes consists of dermal denticles with production of minimal but protein-rich mucus that collectively, influence the attachment and biofilm development of microbes, facilitating a unique epidermal microbiome. Here, we use metagenomics to provide the taxonomic and functional characterization of the epidermal microbiome of the Triakis semifasciata (leopard shark) at three time-points collected across 4 years to identify links between microbial groups and host metabolism. Our aims include (1) describing the variation of microbiome taxa over time and identifying recurrent microbiome members (present across all time-points); (2) investigating the relationship between the recurrent and flexible taxa (those which are not found consistently across time-points); (3) describing the functional compositions of the microbiome which may suggest links with the host metabolism; and (4) identifying whether metabolic processes are shared across microbial genera or are unique to specific taxa. Microbial members of the microbiome showed high similarity between all individuals (Bray–Curtis similarity index = 82.7, where 0 = no overlap, 100 = total overlap) with the relative abundance of those members varying across sampling time-points, suggesting flexibility of taxa in the microbiome. One hundred and eighty-eight genera were identified as recurrent, including Pseudomonas, Erythrobacter, Alcanivorax, Marinobacter, and Sphingopxis being consistently abundant across time-points, while Limnobacter and Xyella exhibited switching patterns with high relative abundance in 2013, Sphingobium and Sphingomona in 2015, and Altermonas, Leeuwenhoekiella, Gramella, and Maribacter in 2017. Of the 188 genera identified as recurrent, the top 19 relatively abundant genera formed three recurrent groups. The microbiome also displayed high functional similarity between individuals (Bray–Curtis similarity index = 97.6) with gene function composition remaining consistent across all time-points. These results show that while the presence of microbial genera exhibits consistency across time-points, their abundances do fluctuate. Microbial functions however remain stable across time-points; thus, we suggest the leopard shark microbiomes exhibit functional redundancy. We show coexistence of microbes hosted in elasmobranch microbiomes that encode genes involved in utilizing nitrogen, but not fixing nitrogen, degrading urea, and resistant to heavy metal.

59 BASIC BIOLOGICAL SCIENCES↗

Microbial Enrichments Contribute to Characterization Of Desert Tortoise Gut Microbiota

Abstract Desert tortoises play ecologically significant roles, including plant seed dispersal and mineral cycling, and yet little is known about microbial members that are critical to their gut and overall health. Tortoises consume recalcitrant plant material, which their gut microbiota degrades and converts into usable metabolites and nutrients for the tortoise. Findings from tortoise gut microbiomes may translate well into biotechnological applications as these microbes have evolved to efficiently degrade recalcitrant substrates and generate useful products. In this study, we cultivated microbial communities from desert tortoise fecal samples following a targeted anaerobic enrichment for microbes involved in deconstruction and utilization of plant biomass. We employed 16S rRNA amplicon sequencing to compare cultivated communities to initial fecal source material and found high abundances of Firmicutes and Bacteroidota typically associated with biomass deconstruction in all cultivated samples. Significantly decreased microbial diversity was observed in the cultivated microbial communities, yet several key taxa thrived in lignocellulose enrichments, includingLachnospiraceaeandEnterococcus. Additionally, cultivated communities produced short-chain fatty acids under anaerobic conditions, and their growth and metabolic output provide evidence of their viability in the initial fecal communities. Overall, this study adds to the limited understanding of reptilian herbivore microbiota, and offers a path towards biotechnological translation based on the ability of the cultivated communities to convert lignocellulose directly to acetate, propionate, and butyrate.

Environmental Sciences & Ecology↗

Omics-driven onboarding of the carotenoid producing red yeast Xanthophyllomyces dendrorhous CBS 6938

Transcriptomics is a powerful approach for functional genomics and systems biology, yet it can also be used for genetic part discovery. Here, we derive constitutive and light-regulated promoters directly from transcriptomics data of the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938 (anamorph Phaffia rhodozyma) and use these promoters with other genetic elements to create a modular synthetic biology parts collection for this organism. X. dendrorhous is currently the sole biotechnologically relevant yeast in the Tremellomycete class-it produces large amounts of astaxanthin, especially under oxidative stress and exposure to light. Thus, we performed transcriptomics on X. dendrorhous under different wavelengths of light (red, green, blue, and ultraviolet) and oxidative stress. Differential gene expression analysis (DGE) revealed that terpenoid biosynthesis was primarily upregulated by light through crtI, while oxidative stress upregulated several genes in the pathway. Further gene ontology (GO) analysis revealed a complex survival response to ultraviolet (UV) where X. dendrorhous upregulates aromatic amino acid and tetraterpenoid biosynthesis and downregulates central carbon metabolism and respiration. The DGE data was also used to identify 26 constitutive and regulated genes, and then, putative promoters for each of the 26 genes were derived from the genome. Simultaneously, a modular cloning system for X. dendrorhous was developed, including integration sites, terminators, selection markers, and reporters. Each of the 26 putative promoters were integrated into the genome and characterized by luciferase assay in the dark and under UV light. The putative constitutive promoters were constitutive in the synthetic genetic context, but so were many of the putative regulated promoters. Notably, one putative promoter, derived from a hypothetical gene, showed ninefold activation upon UV exposure. Thus, this study reveals metabolic pathway regulation and develops a genetic parts collection for X. dendrorhous from transcriptomic data. Therefore, this study demonstrates that combining systems biology and synthetic biology into an omics-to-parts workflow can simultaneously provide useful biological insight and genetic tools for nonconventional microbes, particularly those without a related model organism. This approach can enhance current efforts to engineer diverse microbes.

60 APPLIED LIFE SCIENCES↗

Specialized protist communities on mycorrhizal fungal hyphae

Abstract Arbuscular mycorrhizal (AM) fungi not only play a crucial role in acquiring nutrients for plants but also serve as a habitat for soil microbes. Recent studies observed that AM fungal hyphae are colonized by specific bacterial communities. However, so far it has not been explored whether fungal hyphae and mycorrhizal networks also harbor specific communities of protists, a key group of microbes in the soil microbiome. Here, we characterized protist communities in soil in a compartment with plant roots and on hyphae collected from hyphal compartments without plant roots. We detected specific protist communities on fungal hyphae. Fourteen protistan amplicon sequences variants (ASVs) were significantly associated with fungal hyphae, half of which belonged to the Cercozoa group. This research, for the first-time detected specific protist ASVs directly associated with abundant AM fungus hyphae, highlighting the complexity of the hyphal food web.

Mycology↗

Engineering the cellulolytic extreme thermophile Caldicellulosiruptor bescii to reduce carboxylic acids to alcohols using plant biomass as the energy source

Abstract Caldicellulosiruptor bescii is the most thermophilic cellulolytic organism yet identified (Topt 78 °C). It grows on untreated plant biomass and has an established genetic system thereby making it a promising microbial platform for lignocellulose conversion to bio-products. Here, we investigated the ability of engineered C. bescii to generate alcohols from carboxylic acids. Expression of aldehyde ferredoxin oxidoreductase (aor from Pyrococcus furiosus) and alcohol dehydrogenase (adhA from Thermoanaerobacter sp. X514) enabled C. bescii to generate ethanol from crystalline cellulose and from biomass by reducing the acetate produced by fermentation. Deletion of lactate dehydrogenase in a strain expressing the AOR–Adh pathway increased ethanol production. Engineered strains also converted exogenously supplied organic acids (isobutyrate and n-caproate) to the corresponding alcohol (isobutanol and hexanol) using both crystalline cellulose and switchgrass as sources of reductant for alcohol production. This is the first instance of an acid to alcohol conversion pathway in a cellulolytic microbe.

Biotechnology & Applied Microbiology↗

Low soil phosphorus availability triggers maize growth stage specific rhizosphere processes leading to mineralization of organic P

Phosphorus (P) is one of the essential nutrients for all living organisms. High-quality mineral P fertilizer is a finite resource with only an estimated 80-100 years of reserves remaining globally. Both plants and microbes have developed several mechanisms such as secretion of organic acids, acidification of the rhizosphere, and production of extracellular enzymes to enhance the acquisition of P from soils. Increased activity of phosphatase occurs in response to P deficiency as part of P starvation responses which catalyze the hydrolysis of P o from soil organic matter (SOM) and release inorganic P (P i ). However, the specific interactions between plants and microbial communities in the rhizosphere soil triggered by P deficiency are largely unknown. In a greenhouse study we grew maize plants in low and high P containing soils and examined the role of rhizosphere processes on mineralization of organic P (P o ) in soils with low and high P availability and different P speciation 23 (Po dominated versus P i dominated) and determine its effect on plant growth. Plant biomass was determined and rhizosphere soils, and soil samples from pots with and without plants were collected at key vegetative growth stages (VGS). A variety of biogeochemical parameters (e.g. microbial biomass C and P, potential phosphatase activities, plant biomass P, soil P species etc.) were determined using both conventional (sequential chemical extraction) and advanced (e.g. high resolution mass spectrometry (FTICR-MS) and 3129 P solution NMR) techniques. Here, we used phospholipid fatty acid (PLFA) and DNA based microbial community analysis to track changes in microbial community structure and diversity. Low P availability induced changes in biogeochemical processes and microbial community composition in the rhizosphere soils of plants grown in low P containing soil. At early VGS, the plants in low P soil were visually struggling which correlated with greater rhizosphere potential acid phosphatase activity and a reduction in P o fractions. At late VGS, the plants appeared to recover which correlated with a decrease in Meh (III) extractable P, an increase in microbial biomass C and P and greater total P in the plant biomass (roots, shoots and leaves). In high P containing soil, on the other hand, greater P availability masked these rhizosphere processes. Our results confirmed the degradation of SOM and Po mineralization in rhizosphere soils driven by microbe and plant need for P. P deficiency may have favored the abundance of fungi which utilized easily degradable root exudates for the production of extracellular enzymes responsible for the mineralize of SOM P o . This study highlights the intertwined mechanisms involved, and emphasizes the importance of, P o sources for plant nutrition and survival in marginal soils.

59 BASIC BIOLOGICAL SCIENCES↗

Short-term warming increased soil heterotrophic respiration due to enhanced active microbial community

Soil microbes, particularly active microbes, play a crucial role in conserving soil carbon under climate change, especially in forest ecosystems, constituting over 50% of global soil organic carbon. Nevertheless, the response mechanisms of active microbial community to climate warming and their subsequent impacts on soil heterotrophic respiration (Rh) remain insufficiently understood. To resolve this mechanistic uncertainty, we implemented a 3-year soil translocation experiment to investigate soil warming effects on active microbial community and Rh. We used 16S rRNA gene amplicon sequencing, Metatranscriptomics sequencing, coupled with correlation analyses to explore the linkages between warming-induced shifts in Rh and the active microbial community. Our results demonstrated that warming of soil induced a 55% enhancement in Rh. Concurrently, the Shannon index and Richness of active microbial community increased by 20% and 117%, respectively. Warming significantly altered active microbial community composition, inducing a compositional shift characterized by 0.5-to 2-fold increases in the abundances of Proteobacteria, Chloroflexi, and Actinobacteria. Our study also revealed 92–100% increases in the abundance of C-degradation functional genes encoding starch, cellulose, and lignin decomposition pathways within active microbial community under warming. Statistical analyses identified significant positive correlations between Rh and the abundance of Proteobacteria and Actinobacteria, as well as expression levels of functional genes associated with lignin and cellulose decomposition pathways. Furthermore, our results suggested that short-term warming increased Rh through altering diversity, species composition, and C degradation functional genes of active microbial community providing insights into the influence of microbial communities on soil C-climate feedbacks under climate warming.

Active microbial community↗

Axenisation of oleaginous microalgal cultures via anoxic photosensitisation

Growing interest in sustainable food and biofuel research has necessitated high quality axenic oleaginous microalgal strains. Unfortunately, most strains available in culture banks contain commensal microbes such as bacteria and the default decontamination method involves antibiotic treatment which has begun to exacerbate the emergence of antibiotic resistance. To overcome this problem, anoxic photosensitisation was investigated as an alternate approach. Four oleaginous microalgal species (Tetradesmus obliquus, Desmodesmus armatus, Chlorella vulgaris and Nannochloropsis limnetica) were incubated in varying concentrations of Rose Bengal (0 μM, 1 μM, 3 μM or 9 μM) either in normal (oxic) or anoxic conditions, for 72 h under light (8.85 ± 0.4 W/m 2 ) in a specially designed heterotrophic growth complex (HGC) medium, followed by 72 h in standard Bold's Basal Medium (BBM). Commonly used antibiotics-based protocol was used as the control method. Post treatment, cell numbers and percentage populations were counted with Flow Cytometry, and viability was tested using standard plating methods using BBM and LB. Additionally, the contaminating microbes in the cultures were profiled using 16Ss rRNA sequencing. Anoxic conditions were able to significantly decrease bacterial content, albeit with an equally detrimental effect on the microalgal population. Although the responses differed between the microalgae, anoxic incubation along with Rose Bengal at 3 μM was able to completely decontaminate N. limnetica and C. vulgaris, while D. armatus and T. obliquus could be decontaminated with an additional streak-plating step. None of the cultures could be decontaminated using antibiotics treatment. These results suggest that axenisation of microalgal cultures was largely due to anoxy, that was synergistically enhanced by Rose Bengal at a concentration of ≥3 μM.

59 BASIC BIOLOGICAL SCIENCES↗

Cost impact of hexose-to-pentose sugar ratios for biomanufacturing

Central to the long-term vision for biomanufacturing is the ability to deconstruct plant cell walls to sugars that microbes can convert to products. Aside from glucose, the most abundant sugar in biomass is xylose, a pentose sugar. Industrially relevant microbes have been engineered to co-ferment xylose and glucose. Most nth plant technoeconomic analyses (TEAs) assume similar consumption rates and product yields for both sugars, but in reality, xylose is consumed more slowly. Feedstocks can be selected, or engineered, to alter the glucan-to-xylan ratio (GXR) but no TEAs have quantified the impact of this strategy systematically. This study explores the cost impacts of varying the glucan-to-xylan ratio (GXR) from 1.9 to 6.7 for co-fermenting glucose and xylose to ethanol and bisabolene. The minimum selling prices (MSPs) for both products decrease as the GXR increases, with the largest reductions at shorter residence times. For instance, with an increase in GXR from 1.9 to 6.7, ethanol’s MSP drops by 16 %, 5 %, and 3 % at 24, 72, and 144 h, respectively, while bisabolene’s MSP declines by 23 %, 20 %, and 15 % at 24, 72, and 120 h. Particularly for early-stage commercialization, the results suggest that altering or selecting for feedstocks with higher GXR can minimize capital costs by reducing optimal residence times. Capital-constrained biorefineries operating with shorter residence times can justify paying up to 1.5X to 2X the price for feedstocks with a higher GXR, based on the expected improvements in their product yield and overall process economics.

Delayed xylose utilization↗

Optimizing Cupriavidus necator H16 as a host for aerobic C1 conversion

Biological systems capable of converting CO 2 or CO 2 -derived, single-carbon (C1) compounds can be used to reduce or reverse carbon emissions while establishing a circular bioeconomy to provide sustainable sources of the fuels, foods, and materials humanity relies on. A robust bioeconomy will rely upon a variety of microorganisms capable of assimilating C1 compounds and converting them to valuable products at industrial scale. While anaerobic microbes are ideal hosts for production of short-chain acids and alcohols, microbes capable of aerobic respiration are well suited for biosynthesis of higher molecular weight products. One such organism is the gram-negative soil bacterium Cupriavidus necator, which has been utilized in commercial production of biopolymers for decades. More recently, its capability of robust, aerobic growth on CO 2 has inspired research efforts that have advanced it toward becoming one of the leading bacterial hosts for C1-based biomanufacturing. This review highlights those efforts in the context of the characteristics that have historically made C. necator an excellent host for industrial bioconversion processes: its metabolic versatility, ability to grow rapidly to high cell densities, and genetic amenability.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗