In the OSTI Collections: Microbes for Production and Cleanup
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| Article Acknowledgement: Dr. William N. Watson, Physicist DOE Office of Scientific and Technical Information |
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Like all of our planet’s organisms, microbes, in the process of living, take in various materials and produce others, some for their own use, some as waste. Many products of microbes’ metabolism are useful enough to us that the microbes play significant roles in industrial processes, particularly in the manufacture of food and medicine. But microbes’ roles go far beyond this. As recent research reports and patents demonstrate, people continue to discover more about how to use them.
For example, microbes offer one way to produce nanometer-sized particles of metal oxides—materials in which at least one oxygen atom is chemically combined with one or more metal atoms. Metal oxide particles, which have a variety of uses in magnetic refrigeration devices, optics, and electronics, can be made by purely chemical and mechanical means, but such processes are long, iterative, hard to control, and consume a great deal of energy. Such problems can be avoided by using microbes to make nanoparticles, but depending on the process, the nanoparticles may have to be washed or otherwise separated from microbial matter, thus posing another problem. A recent patent[DOepatents] describes a different way to produce nanoparticles with microbes that involves metal oxides, other materials to be consumed by a culture of microbes, and one or more surfactant compounds[Wikipedia]. The microbes get electrons from the materials they consume and donate them to the metal oxides, which are then separated from the nanoparticles by the surfactants, thus avoiding the need to wash microbial matter off them.
Various ways continue to be found to use different species and strains of microbes, not only to produce certain materials, but to remove other materials from where they’re not wanted. Several recently-explored uses of both types are described below.
Many places where uranium was mined and processed during the Cold War became contaminated with uranium compounds, so the development of techniques for removing those compounds from the local groundwater has been pursued for some years. While earlier reports on microbe-based uranium removal describe explorations of the technique’s feasibility, more recent reports focus on details of how it works and how to improve the basic method.
One research group’s lab experiments with soil bacteria, reported in “Uranium Biomineralization By Natural Microbial Phosphatase Activities in the Subsurface”[SciTech Connect], showed that if a source of organophosphates[Wikipedia] is available, having the bacteria metabolize uranium into uranium phosphate minerals is a viable strategy for immobilizing the uranium. Their studies involved varying the microbes’ conditions in several ways (putting the soil containing them in either aerobic or degassed synthetic groundwater, changing the soil pH[Wikipedia], changing the organophosphates and their concentrations), examining the microbes themselves (including changes in the horizontal transfer of genes[Wikipedia] among some bacteria and in the populations of different species while the microbes metabolize uranium), and determining the stability of the uranium phosphate minerals that the microbes produced. The group’s results aren’t just applicable to uranium cleanup. At the time of their report, they were translating their findings about some of the microbes’ antagonistic properties toward common fungal plant pathogens to support innovative sustainable agricultural practices. “Such technology transfer,” say the researchers, “… will allow farmers to more efficiently make use of phosphate present within fertilizers and sequestered within the soils.”
Another study (“Final Report: Manganese Redox Mediation of UO2 Stability and Uranium Fate in the Subsurface: Molecular and Meter Scale Dynamics” [SciTech Connect]) focused specifically on interactions of uranium with the metallic element manganese, how groundwater constituents affect these interactions, and what happens to the uranium afterwards, as well as whether observed seasonal fluctuations in manganese and the nonmetal selenium are directly linked and whether such linkages can affect the chemical stability of uranium(IV)—i.e., uranium whose atoms contribute, in a certain sense[Wikipedia], 4 electrons each to the other atoms they chemically bind to in molecules. (Uranium(IV) is thus said to have an oxidation number[Wikipedia] of +4.) A third study (“Assessing the Role of Iron Sulfides in the Long Term Sequestration of Uranium by Sulfate-Reducing Bacteria” [SciTech Connect]) aimed to identify how the presence of iron sulfide minerals[Wikipedia] affects the long-term sequestration of uranium(IV). Certain bacteria such as Desulfovibrio vulgaris[Wikipedia] can chemically reduce uranium(VI) dissolved in water to solid compounds containing uranium(IV). Reversal of this reduction—this decrease of oxidation number[Wikipedia]—may be inhibited by iron sulfide. Each of these three reports give many details, learned from different experiments involving different substances and microbes in soil and groundwater, about the many different chemical reactions and metabolic processes that can help or hinder the immobilization of uranium.
Uranium is not the only radioactive substance that needs to be sequestered. Similar microbial processing of technetium[Wikipedia] as well as uranium was addressed by research described in “Molecular Mechanism of Microbial Technetium Reduction Final Report” [SciTech Connect]. This report notes that making technetium insoluble through microbial metabolism provides advantages over removal methods that require soil to be taken offsite for the technetium to be removed by chemical reactions: the offsite methods are often limited by extraction inefficiency, inhibition by chemical reactions that compete with the technetium removal, and production of large volumes of waste. However, the molecular mechanism by which microbes make technetium chemically stable hasn’t been well understood, so the process was examined by mutating technetium-stabilization ability out of Shewanella oneidensis bacteria[Wikipedia] and seeing what they did differently. Results indicated that the different series of chemical compounds that provide pathways for transferring electrons to technetium(VII), nitrate, manganese(III), and uranium(VI) share common structural or regulatory components; the investigators expected to learn even more about the transfer mechanism from newly-determined genomes of a variety of technetium-reducing bacteria.
While this investigation was meant to clarify microbes’ technetium-stabilizing mechanism, another project, reported in “Microbial Activity and Precipitation at Solution-Solution Mixing Zones in Porous Media” [SciTech Connect], aimed to better understand how mixing and delivery of materials in the microbes’ porous habitat affects how efficiently they can immobilize such radioactive contaminants as strontium-90, cobalt-60, and uranium, by characterizing changes in microbe activity, mineral formation, and transport processes in porous media that receive more than one reactive amendment material. In a different investigation (“Redox Interaction of Cytochromes and Bacteria with Oxide Surfaces: Probing Redox-Linked Conformation Change”), the focus was not on particular radioactive contaminants but on understanding how microbial proteins change as they transform them. Findings about protein shape changes and the conditions under which they occur are listed in the project’s Final Technical Report[SciTech Connect].
Radionuclides aren’t the only environmental contaminants to be remediated by microbes. Removal of inorganic salts and various carbon compounds was addressed in work described by three other reports.
Two of these reports are patents. “Microbial fuel cell treatment of fuel process wastewater” [DOepatents], addresses the contamination of water used in drilling for oil and natural gas. These operations use large quantities of water, for example in drilling muds, or for separating bitumen from sand. The water thus used normally contains significant concentrations of high-carbon compounds and inorganic salts. Being unfit for discharge or reuse, the water is usually accumulated near the drilling operation, stored in underground wells, or sent to a treatment facility. None of these options have been cheap. Stored waters are expensive to maintain and typically seep into the environment anyway, while the cost of transporting and treating the water elsewhere can be prohibitive. Cleaning the water onsite has also been tried, but initial efforts have used nonrenewable energy sources intensively, and the membranes employed to remove the contaminants tend to be fouled by suspended matter and crude oil compounds. The invention described by the patent is intended to overcome these problems by contacting the wastewater with a fuel cell anode that contains microbes; the microbes degrade one or more of the undesired carbon compounds and produce electrical energy in the process, which in turn drives a mechanism to reduce the wastewater’s inorganic-salt concentration.
The other patent, “Microbial reductive dehalogenation of vinyl chloride” [DOepatents] addresses the microbial transformation of carcinogenic vinyl chloride[Wikipedia] in groundwater into harmless ethene (C2H4[Wikipedia]). This usually happens under anaerobic conditions when microbes degrade chlorinated solvents into a sequence of materials, including vinyl chloride, that they further degrade until they finally produce ethene. However, at many contaminated sites the degradation stops short of ethene formation, thus causing accumulation of the maximally toxic vinyl chloride. Isolating the genes of a bacterial strain that makes an enzyme for chemically reducing vinyl chloride provides the basis for the invention:
“An isolated strain of bacteria, Dehalococcoides[Wikipedia] sp. strain VS, that metabolizes vinyl chloride is provided; the genetic sequence of the enzyme responsible for vinyl chloride dehalogenation; methods of assessing the capability of endogenous organisms at an environmental site to metabolize vinyl chloride; and a method of using the strains of the invention for bioremediation.”
One other carbon compound that’s problematic in large concentrations is carbon dioxide. As noted in a previous article[OSTI], one way to decrease CO2’s atmospheric concentration is to store it underground. According to “Microbial and Chemical Enhancement of In-Situ Carbon Mineralization in Geological Formation” [SciTech Connect], the best way to keep carbon dioxide underground safely would be easier to determine if we knew more about how it reacts chemically with minerals. As the report’s abstract states, “The ultimate goal of this research project was to develop a microbial and chemical enhancement scheme for in-situ carbon mineralization in geologic formations in order to achieve long-term stability of injected CO2.” Towards this end, interacting systems of CO2, minerals, and brine were systematically studied to determine:
- optimal conditions for microbial production of organic acids
- effects of organic acids on dissolution of magnesium-bearing minerals
- effects of reaction temperature, pH, and geologic-formation pore sizes on mineral carbonates’ crystal structure and chemical composition
- tangible environmental benefit of the in-situ carbon mineral sequestration/sequestering carbon in minerals
The report describes the experiments and computations done to address these questions and presents the resulting data.
Figure 1. Porosity changes during in-situ mineral carbonation as shown and described in “Microbial and Chemical Enhancement of In-Situ Carbon Mineralization in Geological Formation”[SciTech Connect], p. 44: “Enhanced mineral dissolution by injected volatile fatty acids will cause the acceleration of mineral carbonation … [which] may cause porosity decrease or cracking of the rocks. If the porosity of rock decreases with carbonation, it is safe for carbon dioxide storage as mechanical strength increases. If there is rock cracking … the situation is a little more complicated. Poor caprock seal may cause some problems, because it may cause changes in geological formations. However, considering the slow kinetics of in-situ mineral carbonation, which may take years to weather the silicate minerals, it is possible to handle the changes with careful monitoring.”
Getting carbon dioxide from combustion out of the atmosphere isn’t our only problem related to fuel use. A different problem is renewing our supplies of fuel.[OSTI] Fortunately, as we saw above, microbes’ metabolism can not only alter unwanted substances, but can produce wanted ones. Some microbes, in fact, can produce substances that can be used directly as fuel or can help turn other materials into fuels, as several projects show.
Rates at which renewable carbohydrate feedstocks can be converted into oil by microbes have been increased by altering features of the microbes’ metabolism. At any stage of the technology, certain steps of the conversion process will be bottlenecks that limit the oil-production rate. One such bottleneck has been fatty-acid[Wikipedia] synthesis, whose acceleration is impeded by the microbes’ own self-limiting feedback mechanisms: as a microbe accumulates saturated fatty acids, its production of them normally gets inhibited. Another bottleneck is the cost of glucose-based feedstocks. Two patents issued in 2015 address these problems. One answer to the first, described in “Engineered microbes and methods for microbial oil production”[DOepatents], involves modifying microbes so that they combine the production of substances needed to synthesize lipids[Wikipedia] with the sequestration of substances that inhibit fatty-acid production. The other patent, “Engineered microbes and methods for microbial oil overproduction from cellulosic materials”[DOepatents], describes additional measures such as addressing the feedstock-cost problem with modifications to the microbes to let them produce oil from less expensive cellulosic feedstocks.
Figure 2. From the patent “Engineered microbes and methods for microbial oil production”[DOepatents], p. 13: a diagrammatic overview of the principal metabolic pathways for lipid synthesis in the yeast species Yarrowia lipolytica[Wikipedia].
Microbes can thus produce fuel made of hydrogen and carbon, but they aren’t limited to that; alcohol molecules, with the additional element of oxygen, are another microbial product that can be used as fuel. A patent entitled “Conditioning biomass for microbial growth”[DOepatents] notes that while most of the ethanol[Wikipedia] made from biomass is produced by fermenting sugars from corn and sugarcane, the abundance of lignocellulose from forest and farm residues would make it an attractive renewable feedstock if ethanol could be produced from it cheaply. As another report cites[SciTech Connect], if the United States could produce ethanol from the estimated 1.3 billion tons of lignocellulosic biomass[Wikipedia] available each year, the nation could reduce its petroleum consumption by 30%. Things that keep the production expensive include lignocellulose’s being more complex than starches or sugars, the need for pretreatment of woody materials to allow their lignocellulose to be hydrolyzed, and the pretreatment’s byproducts inhibiting ethanol production. The patent describes methods to condition lignocellulose with enzymes that support higher microorganism growth rates, and to culture ethanol-producing microbes in lignocellulosic biomass as well as microbes that are sensitive to reaction-inhibiting compounds.
The report that cited the statistic about 1.3 billion tons a year of lignocellulosic biomass potentially reducing U.S. petroleum consumption by 30%, entitled “Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolysate at high solids loading”[DoE PAGES], describes the breeding of microbe strains—specifically, strains of a particular yeast species—that can produce more ethanol from pretreated lignocellulose. Unlike other yeasts traditionally used in industry, Scheffersomyces stipitis (also called Pichia stipitis)[Wikipedia] can ferment the sugar xylose[Wikipedia], and do so relatively efficiently. Starting with an initial S. stipitis population, many generations of selective breeding resulted in a population that was superior in several respects, including fermentation rates, ethanol tolerance, and ethanol yield, so that a yeast cell density of 0.5 absorbance units, when fermenting hydrolysate in a 5-6 pH medium, could accumulate over 40 grams of ethanol per liter in under 167 hours.
Another report, “Improving microbial biogasoline production in Escherichia coli using tolerance engineering”[DoE PAGES], shows how studying the overexpression of genes that bacterial cells produce more of when isopentenol[PubChem] is present is important for figuring out how to engineer the bacteria to produce short-chain alcohols. (While some strains of E. coli can cause food poisoning, most are harmless, and in fact help their human hosts stay healthy.[Wikipedia; Wikipedia]) The researchers selected 40 such genes, overexpressed them in E. coli, and found that 8 of the genes improved the cells’ tolerance to isopentenol added exogenously. Coexpressing the tolerance-enhancing genes with an isopentenol production pathway showed that 6 of the genes improved E. coli isopentenol production. An expression of one of the genes was, to the researchers’ knowledge, the first example discovered of a transporter protein[Wikipedia] that can be used to improve short-chain alcohol production and “provides a valuable new avenue for host engineering in biogasoline production”.
It would seem that microbes might help solve problems with fuel use at both ends of the overall process: producing hydrocarbons to burn and then sequestering byproducts of their combustion. Actually there’s an additional possibility being explored, namely the use of microbes to produce a different fuel—hydrogen—which has no carbon content. Many different ways of doing this have been considered. Each of three recent reports describes a particular exploration of microbial hydrogen production, each involving a different genus of bacteria.
Since photosynthesizing bacteria and algae that produce hydrogen require an anaerobic (low-oxygen or no-oxygen) environment, their potential for commercially viable hydrogen production is limited. On the other hand, Cyanothece bacteria[Wikipedia] can efficiently make H2 molecules under natural aerobic conditions, as was shown in the “Final Scientific/Technical Report”[SciTech Connect] about the project. The investigators grew cyanobacteria of the strain Cyanothece sp. ATCC 51142 aerobically in an alternating light/dark environment to fit its day/night metabolic cycle, and then took cells from the end of a 12-hour light period and incubated them in airtight vials for another 12 hours under continuous illumination, during which the average H2 production was found to be more than 150 micromoles per milligram of chlorophyll per hour (or over 0.3 milligrams of H2 per milligram of chlorophyll per hour). Growing the Cyanothece cells in the presence of high levels of carbon dioxide or glycerol enhanced the hydrogen production rate. The cells’ enzyme systems for fixing nitrogen from the atmosphere were found to mediate the hydrogen production, and channel all available electrons toward that end when molecular nitrogen is absent: glycerol-supplemented Cyanothece 51142 cells with no nitrogen produced up to 467 micromoles (0.941 milligrams) of H2 per milligram of chlorophyll per hour—an order of magnitude[Wikipedia] faster than other microbes have been found to do anaerobically. The investigators have analyzed Cyanothece 51142’s aerobic H2 production in detail, produced a rich knowledge base for large-scale production, and found that the way carbon dioxide and glycerol—two abundantly available industrial waste products—substantially enhances aerobic H2 production by Cyanothece 51142 suggests that the microbes are a potentially viable means for producing hydrogen as a renewable fuel source.
Photosynthetic H2 production presents its own problems, however. Harvesting the light that powers photosynthesis in pigmented microbes requires their exposure to light over large areas and expensive constant mixing to get them all to the light. This motivated the project reported in “Genetics and Molecular Biology of Hydrogen Metabolism in Sulfate-Reducing Bacteria”[SciTech Connect] to examine fermentative H2 production by Desulfovibrio bacteria. Deleting various genes to determine which were essential for hydrogen production when the bacteria were grown on lactate/sulfate or sulfite media showed that removing any single gene would still let the bacteria produce hydrogen, except for removal of the transmembrane complex DsrMKJOP, which seemed to be essential for hydrogen production in any growth mode, not just growth on lactate/sulfate or sulfite. In a different project, “Biohydrogenesis in the Thermotogales”[SciTech Connect], researchers examined the genetics of a different bacterial genus (Thermotoga[Wikipedia]) to probe the similarities and differences among four species’ hydrogen-production mechanisms. Since “production and consumption of molecular hydrogen drives the physiology and bioenergetics of many microorganisms in hydrothermal environments,” the researchers considered Thermotoga’s potential as models to probe how H2 formation by high-temperature organisms relates to their carbon and energy sources. Many insights gained described in their report have scientific and technological implications.
One more report describes the construction and use of a set of computer models that form “A Systems Biology Platform for Characterizing Regulatory and Metabolic Pathways that Influence and Control Microbial Hydrogen Production” [SciTech Connect]. The developers used their platform to:
- infer the dynamics of how networks of metabolic processes are regulated in a cell,
- study tradeoffs in genetic and nutrient modifications,
- identify regulatory networks of functional genes by calculating variables’ mutual dependence when both are conditioned on a third variable,
- examine how a cell’s hydrogen production can be controlled by altering the strength of proteins that regulate how fast information is transcribed from relevant genes for communication to the rest of the cell,
- analyze experimentally determined phenotypes under different environmental conditions,
- study how microbes are affected by molecules secreted by one species and metabolized by another, as a way to inform the reengineering of microbial metabolism.
The platform can thus help researchers find ways to improve microbial production of hydrogen.
Wikipedia
- Surfactant
- Organophosphate
- pH
- Horizontal gene transfer
- Oxidation state
- Iron sulfide
- Desulfovibrio vulgaris
- Redox
- Technetium
- Shewanella oneidensis
- Vinyl chloride
- Ethylene (or ethene)
- Dehalococcoides
- Fatty acid
- Lipid
- Ethanol
- Yarrowia
- Lignocellulosic biomass
- Pichia stipitis (or Scheffersomyces stipitis)
- Xylose
- Escherichia coli
- Human microbiota
- ATP-binding cassette transporter: Function
- Cyanothece
- Order of magnitude
- Thermotoga
PubChem (open chemistry database of the National Library of Medicine)
Reports available through DOepatents
- “Microbial-mediated method for metal oxide nanoparticle formation” [Metadata]
- “Microbial reductive dehalogenation of vinyl chloride” [Metadata]
- “Microbial fuel cell treatment of fuel process wastewater” [Metadata]
- “Engineered microbes and methods for microbial oil production” [Metadata]
- “Engineered microbes and methods for microbial oil overproduction from cellulosic materials” [Metadata]
- “Conditioning biomass for microbial growth” [Metadata]
Reports available through OSTI’s SciTech Connect
- “Uranium Biomineralization By Natural Microbial Phosphatase Activities in the Subsurface” [Metadata]
- “Final Report: Manganese Redox Mediation of UO2 Stability and Uranium Fate in the Subsurface: Molecular and Meter Scale Dynamics” [Metadata]
- “Assessing the Role of Iron Sulfides in the Long Term Sequestration of Uranium by Sulfate-Reducing Bacteria” [Metadata]
- “Microbial Activity and Precipitation at Solution-Solution Mixing Zones in Porous Media” [Metadata]
- “MOLECULAR MECHANISM OF MICROBIAL TECHNETIUM REDUCTION FINAL REPORT” [Metadata]
- “Final Technical Report” [on “Redox Interaction of Cytochromes and Bacteria with Oxide Surfaces: Probing Redox-Linked Conformation Change”] [Metadata]
- “Microbial and Chemical Enhancement of In-Situ Carbon Mineralization in Geological Formation” [Metadata]
- “A Systems Biology Platform for Characterizing Regulatory and Metabolic Pathways that Influence and Control Microbial Hydrogen Production” [Metadata]
- “Biohydrogenesis in the Thermotogales” [Metadata]
- “Genetics and Molecular Biology of Hydrogen Metabolism in Sulfate-Reducing Bacteria” [Metadata]
- “Final Scientific/Technical Report” [on “Development of Cyanothece as a New Model Organism for Biological Hydrogen Production”] [Metadata]
Reports available through DoE PAGESBeta
- “Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading” [Metadata]
- “Improving microbial biogasoline production in Escherichia coli using tolerance engineering” [Metadata]
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