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DOE OSTI2023

Recalls of tattoo and permanent makeup inks in the United States and a follow-up microbiological survey of inks with a previous recall history

In this study, we collected voluntary recall records of tattoo and permanent makeup ink from the U.S. Food and Drug Administration (US FDA) Enforcement Report Database. The recall records contain information, such as recall date, manufacturer, ink color, reason for recall, and the microorganisms detected from the ink samples. Between 2003 and 2021, a total of 15 voluntary tattoo ink recalls occurred in the U.S. market, involving over 200 tattoo inks marketed by 13 manufacturers and one distributor. Fourteen recalls were due to microbial contamination, and one recall was due to allergic reaction. As follow-up, a microbiological survey of 28 tattoo inks of new batches from seven manufacturers having products that were previously recalled was conducted. Aerobic plate count (APC) and enrichment culture methods based on the FDA’s Bacteriological Analytical Manual (BAM) were used to detect microbial contamination. The results revealed that six out of 28 tattoo inks were contaminated with bacteria and were produced by two manufacturers. The level of microbial contamination was less than 250 CFU/g in three of the tattoo inks and between 1 × 10 3 and 1 × 10 5 CFU/g in the other three inks. Eleven bacterial isolates were identified, including spore-forming Bacillus -related species and potentially pathogenic species. Overall, this study shows that some tattoo ink products produced by manufacturers with a recall history continue to be contaminated with microorganisms. This highlights the need for ongoing monitoring and quality control of such products.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Insight into CAZymes of Alicyclobacillus mali FL18: Characterization of a New Multifunctional GH9 Enzyme

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on β-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

High-Throughput Microbial Community Analyses to Establish a Natural Fungal and Bacterial Consortium from Sewage Sludge Enriched with Three Pharmaceutical Compounds

Emerging and unregulated contaminants end up in soils via stabilized/composted sewage sludges, paired with possible risks associated with the development of microbial resistance to antimicrobial agents or an imbalance in the microbial communities. An enrichment experiment was performed, fortifying the sewage sludge with carbamazepine, ketoprofen and diclofenac as model compounds, with the aim to obtain strains with the capability to transform these pollutants. Culturable microorganisms were obtained at the end of the experiment. Among fungi, Cladosporium cladosporioides, Alternaria alternata and Penicillium raistrickii showed remarkable degradation rates. Population shifts in bacterial and fungal communities were also studied during the selective pressure using Illumina MiSeq. These analyses showed a predominance of Ascomycota (Dothideomycetes and Aspergillaceae) and Actinobacteria and Proteobacteria, suggesting the possibility of selecting native microorganisms to carry out bioremediation processes using tailored techniques.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Mass Spectral Imaging to Map Plant–Microbe Interactions

Plant–microbe interactions are of rising interest in plant sustainability, biomass production, plant biology, and systems biology. These interactions have been a challenge to detect until recent advancements in mass spectrometry imaging. Plants and microbes interact in four main regions within the plant, the rhizosphere, endosphere, phyllosphere, and spermosphere. This mini review covers the challenges within investigations of plant and microbe interactions. We highlight the importance of sample preparation and comparisons among time-of-flight secondary ion mass spectroscopy (ToF-SIMS), matrix-assisted laser desorption/ionization (MALDI), laser desorption ionization (LDI/LDPI), and desorption electrospray ionization (DESI) techniques used for the analysis of these interactions. Using mass spectral imaging (MSI) to study plants and microbes offers advantages in understanding microbe and host interactions at the molecular level with single-cell and community communication information. More research utilizing MSI has emerged in the past several years. We first introduce the principles of major MSI techniques that have been employed in the research of microorganisms. An overview of proper sample preparation methods is offered as a prerequisite for successful MSI analysis. Traditionally, dried or cryogenically prepared, frozen samples have been used; however, they do not provide a true representation of the bacterial biofilms compared to living cell analysis and chemical imaging. New developments such as microfluidic devices that can be used under a vacuum are highly desirable for the application of MSI techniques, such as ToF-SIMS, because they have a subcellular spatial resolution to map and image plant and microbe interactions, including the potential to elucidate metabolic pathways and cell-to-cell interactions. Promising results due to recent MSI advancements in the past five years are selected and highlighted. The latest developments utilizing machine learning are captured as an important outlook for maximal output using MSI to study microorganisms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

The Anaerobic Fungi: Challenges and Opportunities for Industrial Lignocellulosic Biofuel Production

Lignocellulose is a promising feedstock for biofuel production as a renewable, carbohydrate-rich and globally abundant source of biomass. However, challenges faced include environmental and/or financial costs associated with typical lignocellulose pretreatments needed to overcome the natural recalcitrance of the material before conversion to biofuel. Anaerobic fungi are a group of underexplored microorganisms belonging to the early diverging phylum Neocallimastigomycota and are native to the intricately evolved digestive system of mammalian herbivores. Anaerobic fungi have promising potential for application in biofuel production processes due to the combination of their highly effective ability to hydrolyse lignocellulose and capability to convert this substrate to H2 and ethanol. Furthermore, they can produce volatile fatty acid precursors for subsequent biological conversion to H2 or CH4 by other microorganisms. The complex biological characteristics of their natural habitat are described, and these features are contextualised towards the development of suitable industrial systems for in vitro growth. Moreover, progress towards achieving that goal is reviewed in terms of process and genetic engineering. In addition, emerging opportunities are presented for the use of anaerobic fungi for lignocellulose pretreatment; dark fermentation; bioethanol production; and the potential for integration with methanogenesis, microbial electrolysis cells and photofermentation.

pretreatment↗

DOE OSTI2022

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. As a result, these protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Lower functional redundancy in “narrow” than “broad” functions in global soil metagenomics

Understanding the relationship between soil microbial taxonomic compositions and functional profiles is essential for predicting ecosystem functions under various environmental disturbances. However, even though microbial communities are sensitive to disturbance, ecosystem functions remain relatively stable, as soil microbes are likely to be functionally redundant. Microbial functional redundancy may be more associated with “broad” functions carried out by a wide range of microbes than with “narrow” functions in which specific microorganisms specialize. Thus, a comprehensive study to evaluate how microbial taxonomic compositions correlate with broad and narrow functional profiles is necessary. Here, we evaluated soil metagenomes worldwide to assess whether functional and taxonomic diversities differ significantly between the five broad and the five narrow functions that we chose. Our results revealed that, compared with the five broad functions, soil microbes capable of performing the five narrow functions were more taxonomically diverse, and thus their functional diversity was more dependent on taxonomic diversity, implying lower levels of functional redundancy in narrow functions. Co-occurrence networks indicated that microorganisms conducting broad functions were positively related, but microbes specializing in narrow functions were interacting mostly negatively. Our study provides strong evidence to support our hypothesis that functional redundancy is significantly different between broad and narrow functions in soil microbes, as the association of functional diversity with taxonomy was greater in the five narrow than in the five broad functions.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS1971

Microbiologic assay of space hardware.

Review of the procedures used in the microbiological examination of space hardware. The general procedure for enumerating aerobic and anaerobic microorganisms and spores is outlined. Culture media and temperature-time cycles used for incubation are reviewed, along with assay systems designed for the enumeration of aerobic and anaerobic spores. The special problems which are discussed are involved in the precise and accurate enumeration of microorganisms on surfaces and in the neutralization of viable organisms buried inside solid materials that could be released to a planet's surface if the solid should be fractured. Special attention is given to sampling procedures including also the indirect techniques of surface assays of space hardware such as those using detachable or fallout strips. Some data on comparative levels of microbial contamination on lunar and planetary spacecraft are presented.

Favero, M. S.↗

NASA NTRS1972

Detection of metabolically produced labeled gas - The Viking Mars Lander.

A qualitative, nonspecific method will test for life on Mars in 1976 by supplying radioactive substrates to samples of the planetary surface material. If microorganisms are present, they may assimilate one or more of the simple labeled compounds and produce radioactive gas. The compounds have been selected on the basis of biological theory and terrestrial results. The measurement of radioactive gas evolved as a function of time constitutes evidence for life. A control performed on a duplicate, but heat sterilized, sample will confirm the biological nature of the results. The shape of the response curve obtained from the viable sample may provide information on the physiological state and generation period of the organisms. Data obtained from a wide variety of terrestrial soils demonstrate a rapid response and high sensitivity for the experiment. Its ability to make comparative studies of soil microorganisms is also demonstrated.

Levin, G. V.↗

NASA NTRS1972

A stochastic bioburden model for spacecraft sterilization.

Development of a stochastic model of the probability distribution for the random variable representing the number of microorganisms on a surface as a function of time. The first basic principle associated with bioburden estimation is that viable particles are removed from surfaces. The second notion important to the analysis is that microorganisms in environments and on surfaces occur in clumps. The last basic principle relating to bioburden modeling is that viable particles are deposited on a surface. The bioburden on a spacecraft is determined by the amount and kind of control exercised on the spacecraft assembly location, the shedding characteristics of the individuals in the vicinity of the spacecraft, its orientation, the geographical location in which the assembly takes place, and the steps in the assembly procedure. The model presented has many of the features which are desirable for its use in the spacecraft sterilization programs currently being planned by NASA.

Roark, A. L.↗

NASA NTRS1973

Biological effects of fuel and exhaust components from spacecraft descent engines employing hydrazine

The effect of the products of the Viking terminal descent engine fuel upon possible extraterrestrial life at the Martian landing site is examined. The effects of the engine exhaust, the hydrazine fuel, and the breakdown products of the latter on terrestrial microorganisms have been studied. The results indicate that the gaseous exhaust products would probably not be hazardous to microorganisms, but that liquid hydrazine would be lethal.

Lehwalt, M. E.↗

NASA NTRS1971

IMBLMS phase B4, additional tasks 5.0. Microbial identification system

A laboratory study was undertaken to provide simplified procedures leading to the presumptive identification (I/D) of defined microorganisms on-board an orbiting spacecraft. Identifications were to be initiated by nonprofessional bacteriologists, (crew members) on a contingency basis only. Key objectives/constraints for this investigation were as follows:(1) I/D procedures based on limited, defined diagnostic tests, (2) testing oriented about ten selected microorganisms, (3) provide for definitive I/D key and procedures per selected organism, (4) define possible occurrences of false positives for the resulting I/D key by search of the appropriate literature, and (5) evaluation of the I/D key and procedure through a limited field trial on randomly selected subjects using the I/D key.

Source record↗

NASA NTRS1973

Microbiological assay of the Marshall Space Flight Center neutral buoyancy simulator

A neutral buoyancy simulator tank system is described in terms of microbiological and medical safety for astronauts. The system was designed to simulate a gravity-free state for evaluation of orbital operations in a microorganism-free environment. Methods for the identification and elimination of specific microorganisms are dealt with as measures for a pure system of space environment simulation.

Beyerle, F. J.↗

NASA NTRS1973

Biogeochemistry and biogeology research projects

Summaries of work initiated or completed during the year in the areas of biochemistry and biology are reported. Topics include: studying humic substances through geologic time; analyzing the benzene and hexane soluble portions of the benzene-methanol extracts from the same standard suite for hydrocarbons; finding a pyrolytic technique and applying it to single taxa of fossil microorganisms and individual microorganisms; and discovering to what extent humic substances become converted to kerogen and retained in sediments. A list of publications is included.

Cloud, P.↗

NASA NTRS1973

Microbiological profiles of four Apollo spacecraft

The levels and types of microorganisms on various components of four Apollo spacecraft were determined and compared. Although the results showed that the majority of microorganisms isolated were those considered to be indigenous to humans, an increase in organisms associated with soil and dust was noted with each successive Apollo spacecraft.

Puleo, J. R.↗

NASA NTRS1974

Evaluation of membrane filter field monitors for microbiological air sampling

Due to area constraints encountered in assembly and testing areas of spacecraft, the membrane filter field monitor (MF) and the National Aeronautics and Space Administration-accepted Reyniers slit air sampler were compared for recovery of airborne microbial contamination. The intramural air in a microbiological laboratory area and a clean room environment used for the assembly and testing of the Apollo spacecraft was studied. A significantly higher number of microorganisms was recovered by the Reyniers sampler. A high degree of consistency between the two sampling methods was shown by a regression analysis, with a correlation coefficient of 0.93. The MF samplers detected 79% of the concentration measured by the Reyniers slit samplers. The types of microorganisms identified from both sampling methods were similar.

Fields, N. D.↗

NASA NTRS1977

Effect of combined heat and radiation on microbial destruction

A series of experiments at several levels of relative humidity and radiation dose rates was carried out using spores of Bacillus subtilis var. niger to evaluate the effect of heat alone, radiation alone, and a combination of heat and radiation. Combined heat and radiation treatment of microorganisms yields a destruction rate greater than the additive rates of the independent agents. The synergistic mechanism shows a proportional dependency on radiation dose rate, an Arrhenius dependence on temperature, and a dependency on relative humidity. Maximum synergism occurs under conditions where heat and radiation individually destroy microorganisms at approximately equal rates. Larger synergistic advantage is possible at low relative humidities rather than at high relative humidities.

Fisher, D. A.↗

NASA NTRS1977

Microbial metabolic activity in soil as measured by dehydrogenase determinations

The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 C incubation with either glucose or yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.

Casida, L. E., Jr.↗