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NASA NTRS1995

PCR Based Microbial Monitor for Analysis of Recycled Water Aboard the ISSA: Issues and Prospects

The monitoring of spacecraft life support systems for the presence of health threatening microorganisms is paramount for crew well being and successful completion of missions. Development of technology to monitor spacecraft recycled water based on detection and identification of the genetic material of contaminating microorganisms and viruses would be a substantial improvement over current NASA plans to monitor recycled water samples that call for the use of conventional microbiology techniques which are slow, insensitive, and labor intensive. The union of the molecular biology techniques of DNA probe hybridization and polymerase chain reaction (PCR) offers a powerful method for the detection, identification, and quantification of microorganisms and viruses. This technology is theoretically capable of assaying samples in as little as two hours with specificity and sensitivity unmatched by any other method. A major advance in probe-hybridization/PCR has come about in a technology called TaqMan(TM), which was invented by Perkin Elmer. Instrumentation using TaqMan concepts is evolving towards devices that could meet NASA's needs of size, low power use, and simplicity of operation. The chemistry and molecular biology needed to utilize these probe-hybridization/PCR instruments must evolve in parallel with the hardware. The following issues of chemistry and biology must be addressed in developing a monitor: Early in the development of a PCR-based microbial monitor it will be necessary to decide how many and which organisms does the system need the capacity to detect. We propose a set of 17 different tests that would detect groups of bacteria and fungus, as well as specific eukaryotic parasites and viruses; In order to use the great sensitivity of PCR it will be necessary to concentrate water samples using filtration. If a lower limit of detection of 1 microorganism per 100 ml is required then the microbes in a 100 ml sample must be concentrated into a volume that can be added to a PCR assay; There are not likely to be contaminants in ISSA recycled water that would inhibit PCR resulting in false-negative results; The TaqMan PCR product detection system is the most promising method for developing a rapid, highly automated gene-based microbial monitoring system. The method is inherently quantitative. NASA and other government agencies have invested in other technologies that, although potentially could lead to revolutionary advances, are not likely to mature in the next 5 years into working systems; PCR-based methods cannot distinguish between DNA or RNA of a viable microorganism and that of a non-viable organism. This may or may not be an important issue with reclaimed water on the ISSA. The recycling system probably damages the capacity of the genetic material of any bacteria or viruses killed during processing to serve as a template in a PCR desinged to amplify a large segment of DNA (less than 650 base pairs). If necessary, vital dye staining could be used in addition to PCR, to enumerate the viable cells in a water sample; The quality control methods have been developed to insure that PCR's are working properly, and that reactions are not contaminated with PCR carryover products which could lead to the generation of false-positive results; and The sequences of the small rRNA subunit gene for a large number of microorganisms are known, and they consititue the best database for rational development of the oligonucleotide reagents that give PCR its great specificity. From those gene sequences, sets of oligonucleotide primers for PCR and Taqman detection that could be used in a NASA microbial monitor were constructed using computer based methods. In addition to space utilization, a microbial monitior will have tremendous terrestrial applications. Analysis of patient samples for microbial pathogens, testing industrial effluent for biofouling bacteria, and detection biological warfare agents on the battlefield are but a few of the diverse potential uses for this technology. Once fully developed, gene-based microbial monitors will become the fundamental tool in every lab that tests for microbial contaminants, and serve as a powerful weapon in mankind's war with the germ world.

Cassell, Gail H.↗

NASA NTRS1979

Studies on the Mechanisms of Microbial Adaptation to the Physical Environment

The environmental factors which affect humans and other animals also influence the microorganisms which are such an important part of our ecology. Some of the microorganisms are very closely associated with animals, living in the digestive tract and synthesizing essential nutrients for the host. For these microbes, most external physical changes are of little consequence, because they are well shielded by the animals' homeostatic systems. The vast majority of microorganisms, however, live free in nature, especially in the soil and oceans. It has been estimated that the upper 15 cm of a fertile soil may contain over 4000 kg of bacteria and fungi per hectare. These organisms are responsible for degrading the complex molecules of plants and animals when they die, eventually producing simple organics, carbon dioxide, and inorganics, which are then used for the next cycle of plant growth. It is believed that over 90 % of the biologically produced carbon dioxide results from the metabolic activity of bacteria and fungi. In addition to recycling plant nutrients, soil bacteria also provide new nutrients through 'fixation' of atmospheric nitrogen into ammonia and nitrate, the forms which can be used by plants. Microorganisms so have an enormous capacity for detoxifying both natural and man-made poisons. All of these functions of microorganisms are essential to the operation of the material cycles on Earth. This is true of all locations on the planet, regardless of the climate or other environmental factors. In fact, one of the most impressive attributes of microorganisms is their ability to adapt to every stable environment on Earth. These include such extremes as polar regions, hot springs, water saturated with salt, mountain tops, ocean depths, acid and alkaline waters, deserts, intense radioactivity, soil and water contaminated with toxic chemicals or petroleum, and areas devoid of oxygen.

Heinrich, M. R.↗

NASA NTRS2001

Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryo-preserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 T. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica. The microbial extremophiles recovered from permafrost, ice, cold pools and deep sea sediments may provide information relevant to the question of how and where we should search for evidence of extant or extinct microbial life elsewhere in the Cosmos.

Hoover, Richard B.↗

NASA NTRS2002

Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryopreserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 C. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep-sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica. The microbial extremophiles recovered from permafrost, ice, cold pools and deep-sea sediments may provide information relevant to the question of how and where we should search for evidence of extant or extinct microbial life elsewhere in the Cosmos.

Hoover, Richard B.↗

NASA NTRS2005

Microbiological Methodology in Astrobiology

Searching for life in astromaterials to be delivered from the future missions to extraterrestrial bodies is undoubtedly related to studies of the properties and signatures of living microbial cells and microfossils on Earth. As model terrestrial analogs of Martian polar subsurface layers are often regarded the Antarctic glacier and Earth permafrost habitats where alive microbial cells preserved viability for millennia years due to entering the anabiotic state. For the future findings of viable microorganisms in samples from extraterrestrial objects, it is important to use a combined methodology that includes classical microbiological methods, plating onto nutrient media, direct epifluorescence and electron microscopy examinations, detection of the elemental composition of cells, radiolabeling techniques, PCR and FISH methods. Of great importance is to ensure authenticity of microorganisms (if any in studied samples) and to standardize the protocols used to minimize a risk of external contamination. Although the convincing evidence of extraterrestrial microbial life will may come from the discovery of living cells in astromaterials, biomorphs and microfossils must also be regarded as a target in search of life evidence bearing in mind a scenario that alive microorganisms had not be preserved and underwent mineralization. Under the laboratory conditions, processes that accompanied fossilization of cyanobacteria were reconstructed, and artificially produced cyanobacterial stromatolites resembles by their morphological properties those found in natural Earth habitats. Regarding the vital importance of distinguishing between biogenic and abiogenic signatures and between living and fossil microorganisms in analyzed samples, it is worthwhile to use some previously developed approaches based on electron microscopy examinations and analysis of elemental composition of biomorphs in situ and comparison with the analogous data obtained for laboratory microbial cultures and fossilized microorganisms. This communication will be focused on the analysis of our experience in working with ancient microorganisms and fossils and discussion of some issues that are crucial for development of the program for future finding of extraterrestrial life and its evidence.

Abyzov, S. S.↗

NASA NTRS2007

Astrobiological Significance of Microbial Extremophiles

The microflora of the cryosphere of planet Earth provides the best analogs for life forms that might be found in the permafrost or polar ice caps of Mars, near the surface of the cometary nuclei, or in the liquid water beneath and the ice crusts of icy moons of Jupiter and Saturn. The importance of study alkaliphilic microorganisms for astrobiology was enhanced by the findings of abundant carbonates and carbonate globules rimmed with possibly biogenic magnetites in association with the putative microfossils in the ALH84001 meteorite. Although the ALH84001 "nanofossils" were to small and simple to be unambiguously recognized as biogenic, they stimulated Astrobiology research and studies of microbial extremophiles and biomarkers in ancient rocks and meteorites. Recent studies of CI and CM carbonaceous meteorites have resulted in the detection of the well-preserved mineralized remains of coccoidal and filamentous microorganisms in cyanobacterial mats. Energy Dispersive X-ray Analysis has shown anomalous biogenic element ratios clearly indicating they are not recent biological contaminants. This paper reviews microbial extremophiles in context of their significance to Astrobiology. The study of halophilic microorganisms was started from work with saline soils and lakes, and one of the record of good growth for Haloferax mediterranei was shown at 30 percent NaC1. Although alkali-tolerant nitrifying bacteria had previously been reported, the first described alkaliphilic microorganism was the bacterium Streptococcus faecalis. Halophilic and alkaliphilic forms are relevant to conditions that might be found in closed impact basins and craters on Mars filled with evaporite deposits. The first obligately acidophilic bacterium described was Acidithiobacillus ferrooxydans (formally Thiobacillus ferrooxidans). Later thermophilic lithotrophic acidophiles were found, and the hyperacidophilic moderately thermophilic species of the genus Picrophilus were found to grow at negative pH. The epoch of study of thermophilic microorganisms starts with the discovery of Thermus aquaticus, and presently the maximum temperature for growth at 113 C was found for Pyrolobus fumarii. The microorganisms capable of growth at high temperatures and in hyperacidic environments on Earth are good analogs for life that might be able to survive in hot acidic droplets in the upper regimes of the atmosphere of Venus. The study of barophiles was made possible by engineering achievements leading to the development of the submersible crafts used to study the Black Smokers of the Deep-sea Hydrothermal vents. The first described radioresistant bacterium Deinococcus radiodurans can survive ionizing irradiation and other DNA-damaging assaults at doses that are lethal to all other organisms. These microbes are models for life that might endure high radiation environments in the ice near the surface of comets or on the icy moons of Jupiter and Saturn and in the seafloor deep beneath icy crusts Europa and Enceladus. This paper presents ESEM and FESEM images showing intact microbes preserved in the deep ice cores extracted from just above Lake Vostok, Antarctica that are considered analogs for life forms that might survive on comets and icy moons.

Pikuta, Elena V.↗

NASA NTRS2015

Inflight Microbial Monitoring- An Alternative Method to Culture Based Detection Currently Used on the International Space Station

Previous research has shown that potentially destructive microorganisms and human pathogens have been detected on the International Space Station (ISS). The likelihood of introducing new microorganisms occurs with every exchange of crew or addition of equipment or supplies. Microorganisms introduced to the ISS are readily transferred between crew and subsystems (i.e. ECLSS, environmental control and life support systems). Current microbial characterization methods require enrichment of microorganisms and at least a 48-hour incubation time. This increases the microbial load while detecting only a limited number of the total microorganisms. The culture based method detects approximately 1-10% of the total organisms present and provides no identification. To identify and enumerate ISS microbes requires that samples be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, in-flight method of microbial detection, identification, and enumeration is warranted. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganisms at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Inflight Microbial↗

NASA NTRS2015

Inflight Microbial Monitoring-An Alternative Method to Culture Based Detection Currently Used on International Space Station

Previous research has shown that microorganisms and potential human pathogens have been detected on the International Space Station (ISS). The potential to introduce new microorganisms occurs with every exchange of crew or addition of equipment or supplies. Previous research has shown that microorganisms introduced to the ISS are readily transferred between crew and subsystems and back (i.e. ECLSS, environmental control and life support systems). Current microbial characterization methods require enrichment of microorganisms and a 48-hour incubation time. This increases the microbial load while detecting a limited number of microorganisms. The culture based method detects approximately 1-10% of the total organisms present and provides no identification, To identify and enumerate ISS samples requires that samples to be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, in-flight method of microbial detection, identification, and enumeration is warranted. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganism at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Inflight Microbial↗

DOE OSTI2015

Inhibitory effect of high NH{sub 4}{sup +}–N concentration on anaerobic biotreatment of fresh leachate from a municipal solid waste incineration plant

Highlights: • High NH{sub 4}{sup +}–N concentrations inhibit anaerobic treatment of leachate. • Inhibitory effect of NH{sub 4}{sup +}–N concentrations on anaerobic granular sludge is reversible. • High NH{sub 4}{sup +}–N concentrations inhibit bioactivities of microorganisms instead of survival. - Abstract: Fresh leachate from municipal solid waste (MSW) incineration plants generally contains extremely high NH{sub 4}{sup +}–N concentration which could inhibit the bioactivity of microorganisms. The inhibitory effect of high NH{sub 4}{sup +}–N concentration on anaerobic biotreatment of fresh leachate from a MSW incineration plant in China has been investigated in this study. The inhibition processes was studied by both static tests and a laboratory-scale expanded granular sludge bed (EGSB) reactor. The specific methanogenic activity (SMA) of the microorganisms in anaerobic granular sludge was inhibited with the NH{sub 4}{sup +}–N concentration increasing to 1000 mg/L in static tests. As well the chemical oxygen demand (COD) removal efficiency and the methane yield decreased in the EGSB reactor, while the volatile fatty acids (VFAs) accumulated and extracellular polymeric substances (EPS) of the anaerobic granular sludge increased with NH{sub 4}{sup +}–N concentration rising to 1000 mg/L, without any rebounding during 30 days of operation. Decreasing NH{sub 4}{sup +}–N concentration to 500 mg/L in influent, the COD removal efficiency recovered to about 85% after 26 days. 1000 mg/L of NH{sub 4}{sup +}–N in leachate was suggested to be the inhibition threshold in EGSB reactor. High-throughput sequencing results showed little changes in microbial communities of the sludge for a high NH{sub 4}{sup +}–N concentration, indicating that the survival of most microorganisms was not affected under such a condition. It inhibited the bioactivity of the microorganisms, resulting in decrease of the COD removal efficiency.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2021

Colonization of Naive Roots from Populus tremula × alba Involves Successive Waves of Fungi and Bacteria with Different Trophic Abilities

ABSTRACT Through their roots, trees interact with a highly complex community of microorganisms belonging to various trophic guilds and contributing to tree nutrition, development, and protection against stresses. Tree roots select for specific microbial species from the bulk soil communities. The root microbiome formation is a dynamic process, but little is known on how the different microorganisms colonize the roots and how the selection occurs. To decipher whether the final composition of the root microbiome is the product of several waves of colonization by different guilds of microorganisms, we planted sterile rooted cuttings of gray poplar obtained from plantlets propagated in axenic conditions in natural poplar stand soil. We analyzed the root microbiome at different time points between 2 and 50 days of culture by combining high-throughput Illumina MiSeq sequencing of the fungal ribosomal DNA internal transcribed spacer and bacterial 16S rRNA amplicons with confocal laser scanning microscopy observations. The microbial colonization of poplar roots took place in three stages, but bacteria and fungi had different dynamics. Root bacterial communities were clearly different from those in the soil after 2 days of culture. In contrast, if fungi were also already colonizing roots after 2 days, the initial communities were very close to that in the soil and were dominated by saprotrophs. They were slowly replaced by endophytes and ectomycorhizal fungi. The replacement of the most abundant fungal and bacterial community members observed in poplar roots over time suggest potential competition effect between microorganisms and/or a selection by the host. IMPORTANCE The tree root microbiome is composed of a very diverse set of bacterial and fungal communities. These microorganisms have a profound impact on tree growth, development, and protection against different types of stress. They mainly originate from the bulk soil and colonize the root system, which provides a unique nutrient-rich environment for a diverse assemblage of microbial communities. In order to better understand how the tree root microbiome is shaped over time, we observed the composition of root-associated microbial communities of naive plantlets of poplar transferred in natural soil. The composition of the final root microbiome relies on a series of colonization stages characterized by the dominance of different fungal guilds and bacterial community members over time. Our observations suggest an early stabilization of bacterial communities, whereas fungal communities are established following a more gradual pattern.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2010

Characterization of Microbial Communities in Subsurface Nuclear Blast Cavities of the Nevada Test Site

This U.S. Department of Energy (DOE) Environmental Remediation Sciences Project (ERSP) was designed to test fundamental hypotheses concerning the existence and nature of indigenous microbial populations of Nevada Test Site subsurface nuclear test/detonation cavities. Now called Subsurface Biogeochemical Research (SBR), this program’s Exploratory Research (ER) element, which funded this research, is designed to support high risk, high potential reward projects. Here, five cavities (GASCON, CHANCELLOR, NASH, ALEMAN, and ALMENDRO) and one tunnel (U12N) were sampled using bailers or pumps. Molecular and cultivation-based techniques revealed bacterial signatures at five sites (CHANCELLOR may be lifeless). SSU rRNA gene libraries contained diverse and divergent microbial sequences affiliated with known metal- and sulfur-cycling microorganisms, organic compound degraders, microorganisms from deep mines, and bacteria involved in selenate reduction and arsenite oxidation. Close relatives of Desulforudis audaxviator, a microorganism thought to subsist in the terrestrial deep subsurface on H2 and SO42- produced by radiochemical reactions, was detected in the tunnel waters. NTS-specific media formulations were used to culture and quantify nitrate-, sulfate-, iron-reducing, fermentative, and methanogenic microorganisms. Given that redox manipulations mediated by microorganisms can impact the mobility of DOE contaminants, our results should have implications for management strategies at this and other DOE sites.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2017

Lignocellulose deconstruction in the biosphere

Microorganisms have evolved different and yet complementary mechanisms to degrade biomass in the biosphere. The chemical biology of lignocellulose deconstruction is a complex and intricate process that appears to vary in response to specific ecosystems. These microorganisms rely on simple to complex arrangements of glycoside hydrolases to conduct most of these polysaccharide depolymerization reactions and also, as discovered more recently, oxidative mechanisms via lytic polysaccharide monooxygenases or non-enzymatic Fenton reactions which are used to enhance deconstruction. It is now clear that these deconstruction mechanisms are often more efficient in the presence of the microorganisms. In general, a major fraction of the total plant biomass deconstruction in the biosphere results from the action of various microorganisms, primarily aerobic bacteria and fungi, as well as a variety of anaerobic bacteria. Beyond carbon recycling, specialized microorganisms interact with plants to manage nitrogen in the biosphere. Understanding the interplay between these organisms within or across ecosystems is crucial to further our grasp of chemical recycling in the biosphere and also enables optimization of the burgeoning plant-based bioeconomy.

09 BIOMASS FUELS↗

DOE OSTI2011

Live wires: direct extracellular electron exchange for bioenergy and the bioremediation of energy-related contamination

Microorganisms that can form direct electrical connections with insoluble minerals, electrodes, or other microorganisms can play an important role in some traditional as well as novel bioenergy strategies and can be helpful in the remediation of environmental contamination resulting from the use of more traditional energy sources. The surprising discovery that microorganisms in the genus Geobacter are capable of forming highly conductive networks of filaments that transfer electrons along their length with organic metallic-like conductivity, rather than traditional molecule to molecule electron exchange, provides an explanation for the ability of Geobacter species to grow in subsurface environments with insoluble Fe(III) oxides as the electron acceptor, and effectively remediate groundwater contaminated with hydrocarbon fuels or uranium and similar contaminants associated with the mining and processing of nuclear fuel. A similar organic metallic-like conductivity may be an important mechanism for microorganisms to exchange electrons in syntrophic associations, such as those responsible for the conversion of organic wastes to methane in anaerobic digesters, a proven bioenergy technology. Biofilms with conductivities rivaling those of synthetic polymers help Geobacter species generate the high current densities in microbial fuel cells producing electric current from organic compounds. Electron transfer in the reverse direction, i.e. from electrodes to microbes, is the basis for microbial electrosynthesis, in which microorganisms reduce carbon dioxide to fuels and other useful organic compounds with solar energy in a form of artificial photosynthesis that is more efficient and avoids many of the environmental sustainability concerns associated with biomass-based bioenergy strategies. The ability of Geobacter species to produce highly conductive electronic networks that function in water opens new possibilities in the emerging field of bioelectronics.

Lovley, DR↗

DOE OSTI2020

Pore-scale view of microbial turnover: combining 14 C imaging, μCT, and zymography after adding soluble carbon to soil pores of specific sizes

The location of microorganisms and substrates within soil pore networks plays a crucial role for organic carbon (C) processing, its microbial utilization and turnover, and has direct consequences for C and nutrient cycling. An optimal approach to quantify responses to new C inputs from microorganisms residing in specific pores is the addition of new C to pores of target sizes in undisturbed soil cores. We used the matric potential approach to add 14 C labeled glucose to small (< 40 μm, root free) or large (60-180 μm, potentially inhabited by roots) pores of undisturbed soil cores. Localization of glucose-derived C via 14 C imaging was related to pore size distributions and connectivity, characterized via X-ray computed micro-tomography (μCT), and to β-glucosidase activity, characterized via zymography. After two-week incubations, 1.3 times more glucose was mineralized ( 14 CO 2 ) when it was added to the large pores, however more 14 C remained in microbial biomass when glucose was added to the small pores. Consequently, while utilizing the same amounts of easily available C, the microorganisms localized in the large pores had faster turnover compared to microorganisms in small pores. Stronger associations between β-glucosidase activity and glucose-derived C were observed when glucose was added to the large pores. We conclude that 1) the matric potential approach allows placing, albeit not exactly, soluble substrates into pores of target diameter range, and 2) microorganisms localized in large pores respond to new C inputs with faster turnover, greater growth, and more intensive enzyme production compared to those inhabiting the small pores.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2017

Conversion of Amazon rainforest to agriculture alters community traits of methane-cycling organisms

Land use change is one of the greatest environmental impacts worldwide, especially to tropical forests. The Amazon rainforest has been subject to particularly high rates of land use change, primarily to cattle pasture. A commonly observed response to cattle pasture establishment in the Amazon is the conversion of soil from a methane sink in rainforest, to a methane source in pasture. However, it is not known how the microorganisms that mediate methane flux are altered by land use change. Here, we use the deepest metagenomic sequencing of Amazonian soil to date to investigate differences in methane-cycling microorganisms and their traits across rainforest and cattle pasture soils. We found that methane-cycling microorganisms responded to land use change, with the strongest responses exhibited by methane-consuming, rather than methane-producing, microorganisms. These responses included a reduction in the relative abundance of methanotrophs and a significant decrease in the abundance of genes encoding particulate methane monooxygenase. We also observed compositional changes to methanotroph and methanogen communities as well as changes to methanotroph life history strategies. Our observations suggest that methane-cycling microorganisms are vulnerable to land use change, and this vulnerability may underlie the response of methane flux to land use change in Amazon soils.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2012

Engineering a Hyperthermophilic Archaeon for Temperature-Dependent Product Formation

Microorganisms growing near the boiling point have enormous biotechnological potential but only recently have molecular engineering tools become available for them. We have engineered the hyperthermophilic archaeon Pyrococcus furiosus, which grows optimally at 100 degrees C, to switch its end products of fermentation in a temperature-controlled fashion without the need for chemical inducers. The recombinant strain (LAC) expresses a gene (ldh) encoding lactate dehydrogenase from the moderately thermophilic Caldicellulosiruptor bescii (optimal growth temperature [T-opt] of 78 degrees C) controlled by a "cold shock" promoter that is upregulated when cells are transferred from 98 degrees C to 72 degrees C. At 98 degrees C, the LAC strain fermented sugar to produce acetate and hydrogen as end products, and lactate was not detected. When the LAC strain was grown at 72 degrees C, up to 3 mM lactate was produced instead. Expression of a gene from a moderately thermophilic bacterium in a hyperthermophilic archaeon at temperatures at which the hyperthermophile has low metabolic activity provides a new perspective to engineering microorganisms for bioproduct and biofuel formation. IMPORTANCE Extremely thermostable enzymes from microorganisms that grow near or above the boiling point of water are already used in biotechnology. However, the use of hyperthermophilic microorganisms themselves for biotechnological applications has been limited by the lack of their genetic accessibility. Recently, a genetic system for Pyrococcus furiosus, which grows optimally near 100 degrees C, was developed in our laboratory. In this study, we present the first heterologous protein expression system for a microorganism that grows optimally at 100 degrees C, a first step towards the potential expression of genes involved in biomass degradation or biofuel production in hyperthermophiles. Moreover, we developed the first system for specific gene induction in P. furiosus. As the cold shock promoter for protein expression used in this study is activated at suboptimal growth temperatures of P. furiosus, it is a powerful genetic tool for protein expression with minimal interference of the host's metabolism and without the need for chemical inducers.

Basen, M↗

DOE OSTI2015

Metaproteomics Identifies the Protein Machinery Involved in Metal and Radionuclide Reduction in Subsurface Microbiomes and Elucidates Mechanisms and U(VI) Reduction Immobilization

The overall goal for this funded project was to develop and exploit environmental metaproteomics tools to identify biomarkers for monitoring microbial activity affecting U speciation at U-contaminated sites, correlate metaproteomics profiles with geochemical parameters and U(VI) reduction activity (or lack thereof), elucidate mechanisms contributing to U(VI) reduction, and provide remediation project managers with additional information to make science-based site management decisions for achieving cleanup goals more efficiently. Although significant progress has been made in elucidating the microbiology contribution to metal and radionuclide reduction, the cellular components, pathway(s), and mechanisms involved in U trans-formation remain poorly understood. Recent advances in (meta)proteomics technology enable detailed studies of complex samples, including environmental samples, which differ between sites and even show considerable variability within the same site (e.g., the Oak Ridge IFRC site). Additionally, site-specific geochemical conditions affect microbial activity and function, suggesting generalized assessment and interpretations may not suffice. This research effort integrated current understanding of the microbiology and biochemistry of U(VI) reduction and capitalize on advances in proteomics technology made over the past few years. Field-related analyses used Oak Ridge IFRC field ground water samples from locations where slow-release substrate biostimulation has been implemented to accelerate in situ U(VI) reduction rates. Our overarching hypothesis was that the metabolic signature in environmental samples, as deciphered by the metaproteome measurements, would show a relationship with U(VI) reduction activity. Since metaproteomic and metagenomic characterizations were computationally challenging and time-consuming, we used a tiered approach that combines database mining, controlled laboratory studies, U(VI) reduction activity measurements, phylogenetic analyses, and gene expression studies to support the metaproteomics characterizations. Growth experiments of target microorganisms (Anaeromyxobacter, Shewanella, Geobacter) revealed tremendous respiratory versatility, as evidenced by the ability to utilize a range of electron donors (e.g. acetate, hydrogen, pyruvate, lactate, succinate, formate) and electron acceptors (e.g. nitrate, fumarate, halogenated phenols, ferric iron, nitrous oxide, etc.). In particular, the dissimilatory metabolic reduction of metals, including radionuclides, by target microorganisms spurred interest for in situ bioremediation of contaminated soils and sediments. Distinct c-type cytochrome expression patterns were observed in target microorganisms grown with the different electron acceptors. For each target microorganism, the core proteome covered almost all metabolic pathways represented by their corresponding pan-proteomes. Unique proteins were detected for each target microorganism, and their expression and possible functionalities were linked to specific growth conditions through proteomics measurements. Optimization of the proteomic tools included in-depth comprehensive metagenomic and metaproteomic analyses on a limited number of samples. The optimized metaproteomic analyses were then applied to Oak Ridge IFRC field samples from the slow-release substrate biostimulation. Metaproteomic analysis and pathway mapping results demonstrated the distinct effects of metal and non-metal growth conditions on the proteome expression. With these metaproteomic tools, we identified which previously hypothetical metabolic pathways were active during the analyzed time points of the slow release substrate biostimulation. Thus, we demonstrated the utility of these tools for site assessment, efficient implementation of bioremediation and long-term monitoring. This research of detailed protein analysis linked with metal reduction activity did (1) show that c-type cytochrome isoforms, previously associated with radionuclide reduction activity, are suitable biomarkers, (2) identify new biomarker targets for site assessment and bioremediation monitoring, and (3) provide new information about specific proteins and mechanisms involved in U(VI) reduction and immobilization. This expanded metagenomic and metaproteomic toolbox contributed to implementing science-driven site management with broad benefits to the DOE mission.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS1982

Apparatus and process for microbial detection and enumeration

An apparatus and process for detecting and enumerating specific microorganisms from large volume samples containing small numbers of the microorganisms is presented. The large volume samples are filtered through a membrane filter to concentrate the microorganisms. The filter is positioned between two absorbent pads and previously moistened with a growth medium for the microorganisms. A pair of electrodes are disposed against the filter and the pad electrode filter assembly is retained within a petri dish by retainer ring. The cover is positioned on base of petri dish and sealed at the edges by a parafilm seal prior to being electrically connected via connectors to a strip chart recorder for detecting and enumerating the microorganisms collected on filter.

Wilkins, J. R.↗