BOARD OF GOVERNORS, AMERICAN ACADEMY OF MICROBIOLOGY

R. John Collier, Ph.D. (Chair), Harvard University Medical School

Kenneth I. Berns, M.D., Ph.D., University of Florida Genetics Institute

Arnold L. Demain, Ph.D., Drew University

E. Peter Greenberg, Ph.D., University of Washington

Carol A. Gross, Ph.D., University of California, San Francisco

J. Michael Miller, Ph.D., Centers for Disease Control and Prevention

Stephen A. Morse, Ph.D., Centers for Disease Control and Prevention

Harriet L. Robinson, Ph.D., Emory University

George F. Sprague, Jr., Ph.D., University of Oregon

David A. Stahl, Ph.D., University of Washington

Judy A. Wall, Ph.D., University of Missouri-Columbia

COLLOQUIUM STEERING COMMITTEE

Judy A. Wall, Ph.D., (Chair), University of Missouri-Columbia

Lonnie O. Ingram, Ph.D., (Co-Chair), University of Florida

Jean E. Brenchley, Ph.D., The Pennsylvania State University

Caroline S. Harwood, Ph.D., University of Washington

Lee Lynd, D.E., Dartmouth College

Carol A. Colgan, Director, American Academy of Microbiology

SPONSOR REPRESENTATIVES

Parag R. Chitnis, Ph.D., National Science Foundation

Michael L. Knotek, Ph.D., U.S. Department of Energy

Sherry Tove, Ph.D., U.S. Army Research Office

Sharlene Weatherwax, Ph.D., U.S. Department of Energy

COLLOQUIUM PARTICIPANTS

Birgitte Ahring, Ph.D., Technical University of Denmark

Frances Arnold, Ph.D., California Institute of Technology

Hans Blaschek, Ph.D., University of Illinois

Brian H. Davison, Ph.D., Oak Ridge National Laboratory

Arnold L. Demain, Ph.D., Drew University

Roy Doi, Ph.D., University of California, Davis

Douglas Eveleigh, Ph.D., Rutgers University

J. Gregory Ferry, Ph.D., The Pennsylvania State University

Yuri A. Gorby, Ph.D., Pacific Northwest National Laboratory

Gerhard Gottschalk, Sc.D., Institut fur Mikrobiologie und Genetics, Goettingen, Germany

Barbel Hahn-Hagerdal, Ph.D., Lund University, Lund, Sweden

Mark T. Holtzapple, Ph.D., Texas A&M University

Peter Lindblad, Ph.D., Uppsala University, Villavagen, Sweden

Derek Lovley, Ph.D., University of Massachusetts-Amherst

Paul W. Ludden, Ph.D., University of California, Berkeley

Julie A. Maupin-Furlow, Ph.D., University of Florida

Kenneth H. Nealson, Ph.D., University of Southern California

Dianne K. Newman, Ph.D., California Institute of Technology

Nancy Nichols, Ph.D., U.S. Department of Agriculture, Peoria, Illinois

John Reeve, Ph.D., Ohio State University

John Saddler, Ph.D., University of British Columbia, Vancouver, Canada

Bernhard Schink, Sc.D., University of Konstanz, Germany

Michael Seibert, Ph.D., National Renewal Energy Laboratory, Golden, Colorado

Alfred Spormann, Ph.D., Stanford University

Joseph M. Suflita, Ph.D., University of Oklahoma

Robert F. Tabita, Ph.D., Ohio State University

Larry P. Walker, Ph.D., Cornell University

Paul J. Weimer, Ph.D., University of Wisconsin, Madison

William Whitman, Ph.D., University of Georgia

Ann C. Wilkie, Ph.D., University of Florida

PROS AND CONS OF MICROBIAL ENERGY CONVERSION

Microbial energy conversion has several advantages over petroleum- and natural gas-based energy. Primary among these benefits is the fact that the processes involved in microbial energy transformations are more environmentally friendly than fossil fuel technologies. For example, microbial energy modes do not involve the use or production of hazardous materials. More importantly, all of the microbial energy transformation processes described here, except for photosynthetic hydrogen generation, involve the conversion of biomass into chemical or electrical energy. Biomass conversion is a “carbon-neutral” process, meaning potentially it neither increases nor decreases the total amount of carbon dioxide (an important greenhouse gas) in the atmosphere.

Microorganisms are also capable of selecting their substrate from among a complex mixture of chemicals, an ability that minimizes the need to purify or refine the raw materials to separate substrates from non-substrates. Finally, microbial energy technologies can be used to produce energy locally, close to where the energy is needed. “Distributed” energy systems like these can minimize the cost of energy transfer, a big consideration where energy needs are small.

Although biology is to thank for many of the advantages of using microorganisms in energy conversion, biology is also behind many of the drawbacks to using these technologies. Many of the microbial strains and enzymes used to produce fuels, for example, are sensitive to the presence of their products, so there is often a need for physical separation of intermediates or products from the reaction center in order to drive the process forward. Hydrogenases and nitrogenases, which produce hydrogen (a potentially very powerful biofuel), are extremely sensitive to oxygen. Hence, hydrogen-generating bioreactors must be kept under strictly anoxic conditions, a state that is not always easy to maintain.

MEETING THE WORLD'S ENERGY NEEDS

Although the field is promising, microbial energy conversion cannot possibly meet all of the world's diverse energy needs. Different applications can call for radically different solutions. Microbial sources of energy may fit into some niches (depending on the amount of energy needed and the locale), but it is necessary to make a wide variety of sound energy technologies available to decision makers.

Currently, the choices for providing energy are limited. Policy makers and the research community must begin to pursue a broader array of potential energy technologies. A diverse energy portfolio that includes an assortment of microbial energy choices will allow communities and consumers to select the best energy solution for their own particular needs.

Funding agencies and governments alike need to prepare for future energy needs by investing both in the microbial energy technologies that work today and in the untested technologies that will serve the world's needs tomorrow. More mature bio-processes, such as ethanol production from starchy materials and methane from waste digestors, will find applications in the short term. However, innovative techniques for liquid fuel or biohydrogen production are among the longer term possibilities that should also be vigorously explored, starting now.

BIOETHANOL

When manufactured from crop sources, like corn or wood, ethanol is commonly referred to as “bioethanol.” Theoretically, bioethanol production should be able to yield 0.5 g of ethanol per gram of raw biomass, which translates into an energy recovery of approximately 90%. Current technologies recover roughly 60% of the energy in raw biomass.

Bioethanol is flexible; it can be manufactured from any of a number of different substrates (including starch and lignocellu-lose), by a selection of different microorganisms, and in a variety of different engineered circumstances. Corn is a common substrate for bioethanol manufacture because the process is relatively free of technical obstacles and because, in the U.S. at least, the use of corn as a substrate for energy production is subsidized by the federal government. Microorganisms are involved in the transformation of corn to bioethanol in two ways: they catalyze the hydrolysis of starches using amylases and amyloglucosidases, and they ferment the resulting sugars to bioethanol. The fermentation step is generally carried out by yeast, but certain strains of bacteria, including Zymomonas mobilis and recombinant strains of Escherichia coli and Klebsiella oxytoca, are also capable of producing high yields of bioethanol.

Lignocellulose includes such diverse sources as switchgrass, corn stalks, and wood chips. Fungal enzymes and fermentative yeasts are then used to transform lignocellulose first to sugars and then to bioethanol. The low lignocellulose reactivity limits the production of bioethanol, and as a result, the concentration of bioethanol in a lignocellulose reactor does not reach the high concentrations seen in corn reactors. Hence, bioethanol-producing microbes in corn-based reactor systems must be tolerant of higher ethanol concentrations than microbes in lignocellulose reactors.

Consolidated bioprocessing (CBP) systems, in which anaerobic bacteria produce their own hydrolytic enzymes and ferment the polysaccharide oligomers, also can be used to produce bioethanol. CPB systems bypass the enzyme extraction step, thereby theoretically enhancing the efficiency of bioethanol production. Recent successes in understanding the genetics of the cellulose fermenter Clostridium thermocellum, which is often used in CPB systems, are encouraging. Low yield, low bioethanol concentration, low volumetric productivity, low product tolerance in the bacteria, growth medium requirements, and intractable genetics still pose difficulties in applying CPB, however.

The MixAlco process¹ is another promising technology for mixed alcohol production. MixAlco may be used to convert any biodegradable material into a mixture of carboxylate salts, which may be chemically converted to primary or secondary alcohols and other valuable products, including carboxylic acids, ketones, esters, ethers, and aldehydes. For any given product, the molecular weight distribution may be influenced by adjusting the reaction parameters. The substrate, which can range from municipal waste to cattle manure to energy crops, is pretreated with lime and oxygen to increase its digestibility and then fed into a mixed culture fermenter. Currently, the pilot plant is digesting paper waste and manures, but pretreatment can enable the use of more recalcitrant biomass. The solids retention time for MixAlco, a measure of how long it takes for a material to be completely digested, varies from 20 days to three months. The system has been operated at a pilot scale (8,000 liters), and it is claimed that the process can generate roughly 86 gallons of mixed secondary alcohols or 125 gallons of mixed primary alcohols per dry ton of biomass. Under anaerobic conditions, the products of MixAlco are stable and may be stored or shipped to a central processing facility. The culture used for fermentation has yet to be thoroughly described, but it is known from clone libraries that it is composed of roughly 20 species, half of which have been previously described.

In a method known as the Gaddy process (patented process developed by James Gaddy of Fayetteville, Arkansas), carbon monoxide gas derived from biomass is converted to bioethanol by a proprietary culture of microorganisms, in which Clostridium ljungdahlii, plays a central role. The cost of introducing gas into the liquid culture and extracting the bioethanol is high, but as refinements have been implemented, the process has become more cost-effective and may be as low as $0.80 per gallon of bioethanol.

Bioethanol is flexible: it can be manufactured from any of a number of different substrates (including starch and lignocellulose), by a selection of different microorganisms, and in a variety of different engineered circumstances.

Disadvantages and Technical Difficulties

Although bioethanol production has been successful in a number of different respects, technical issues have arisen in efforts to produce bioethanol at a commercial scale. Unsatisfactory yield has been one problem. Efforts should be made to change the efficiency of the bioethanol reaction in order to improve yields.

Using solid substrates in converting lignocellulose to sugars poses another dilemma in bioethanol production. The substrate availability in solid materials is quite low in comparison to liquid materials, a limitation that constricts the rates and yields. It may be possible to use decrystallizing enzymes or cellulosomes (enzyme complexes that degrade cellulose) to open the structures of the organic material and make the cellulose more available to microorganisms.

(http://www.eia.doe.gov/kids/energyfacts/sources/non-renewable/oil.html#How%20formed)

Cellulose and lignocellulose are in much greater supply than starch and sugars and are, therefore, preferred substrates for ethanol production. However, producing ethanol from cellulose and lignocellulose is comparatively difficult and expensive. A number of challenges must be overcome in order to make the conversion of cellulose and lignocellulose to ethanol more cost-competitive, including:

• Optimizing different biomass pretreatment processes for different substrates,
• Engineering more efficient enzymes for lignocellulose and hemicellulose degradation,
• Developing more efficient fermenting organisms, and
• Reducing the cost of producing cellulose- and lignocellulose-degrading enzymes.

The expense of enzyme production is the biggest economic barrier to lignocellulose conversion to bioethanol. Fungal cellulases and beta-glucosidases produced in separate aerobic reactors can be extracted in very high yields, but because these enzymes have low specific activities, they must be used in large quantities to achieve lignocellulose conversion. A great deal of effort and funding has been directed toward improving the specific activity of these enzymes in order to increase the cost-effectiveness of lignocellulose conversion to bioethanol, but this work has failed to achieve significant success.

Pretreated wood may contain phenolics, furan compounds, and short-chain fatty acids that are inhibitory to fermenting microorganisms. Hence, wood products usually are subjected to a costly detoxification step prior to fermentation. Microorganisms that can tolerate these inhibitory compounds do exist, however, so it should be possible to avoid having to remove inhibitors.

Ethanol adsorbs water readily and cannot be shipped through common-carrier pipelines, which inevitably contain water. Ethanol currently is shipped by truck or rail and splash blended at the terminal, which adds a great deal of cost. Higher molecular weight alcohols, such as butanol, are more compatible with the existing liquid fuel infrastructure.

HYDROGEN

The greatest advantage of hydrogen over other microbially produced energy carriers lies in its flexibility; it can be produced in a number of different ways by many different organisms. Storage and distribution of hydrogen are problematic, however. Development of a liquid fuel with a high hydrogen content could solve some storage and transport issues, allowing the use of hydrogen to mesh with the distribution technology and infrastructure already in place today.

A variety of approaches should be explored to advance the long-term goal of efficient microbial hydrogen production. Hydrogen production by cyanobacteria, anoxygenic photosynthetic bacteria, and green algae need to be explored, as does the potential for hydrogen production in organisms yet to be discovered. Metabolic engineering of hydrogen production in known microbes should also be researched. New genomics and genetic exchange tools are needed to fully realize the possibilities of engineering and optimizing hydrogen production in microorganisms.

Coupling Oxygenic Photosynthesis and Hydrogenases

With the aid of the sun and CO₂, cyanobacteria, microalgae, and macroalgae all produce biomass by the metabolic pathway known as “photosystem II.” In the absence of CO₂, some of these photosynthetic organisms can produce hydrogen. Employing oxygenic photosynthesis to produce hydrogen on a commercial scale has great potential in the long term, but there are also technical difficulties and disadvantages to using the process.

Development of a liquid fuel with a high hydrogen content could solve some storage and transport issues, allowing the use of hydrogen to mesh with the distribution technology and infrastructure already in place today.

Advantages

The resources necessary to make hydrogen phototrophically, namely water and sunlight, are virtually unlimited. Moreover, the process does not rely on the use of biomass, reduced sulfur compounds, or reduced iron, as do other modes of microbial hydrogen production. Aquatic phototrophic microorganisms are the preferred organisms for making hydrogen, since they produce a lignin-free biomass that is easily digested under anaerobic conditions, making waste handling a relatively simple matter. Since exploration of this field has begun relatively recently, opportunities for improving phototrophic hydrogen production are numerous, and there is the potential to make substantial gains in efficiency and yield.

Disadvantages and Technical Difficulties

Some technical difficulties with using oxygenic phototrophic hydrogen production as a mode of energy conversion include inefficiencies of light capture in bioreactor systems and inefficiencies in the water splitting/manganese chemistry involved. Hydrogen-producing phototrophs currently extract light at about 0.1% efficiency in bioreactors. Methods to enhance penetration of solar energy to the photosynthetic machinery of the cell are needed to improve bioreactor efficiency and to facilitate expansion of these systems for industrial bioreactors. Film covers for photobioreactors that can cover thousands of square kilometers are durable, light-transparent, resistant to ultraviolet radiation, and gas impermeable are also needed.

Research Needs

Hydrogenases, the proteins that generate hydrogen, are inhibited by even very small quantities of hydrogen, so hydrogen must be removed from the system as soon as it is created in order for the process to continue efficiently. New and improved techniques for accomplishing hydrogen removal are needed and may require the involvement of materials scientists. Hydrogenases are also damaged by the other product of oxygenic photosynthesis, oxygen. It may be possible to optimize hydrogen production by employing naturally oxygen-tolerant hydrogenases like iron hydrogenases for the purpose, but further work is required to understand these enzymes and to enhance their electron turnover. The products of phototrophic hydrogen evolution, hydrogen and oxygen, are explosive. The design of the reactors used for this purpose needs to be improved to overcome these inefficiencies and hazards.

Photobioreactor development is also needed. New bioreactors should be designed modularly to enable creation of other value-added products by the photosystem. For example, a well-designed photobioreactor could be made into a microbial fuel cell, directing electrons from the photosystem to make electricity instead of hydrogen.

(“Worldwide Look at Reserves and Production,” Oil and Gas Journal, vol. 47 (Dec. 19, 2005), pp. 24-25.)

(History: Energy Information Administration, International Energy Annual (IEA) 2003 (May-July 2005), website http://www.eia.doe.gov/iea/. Projections: IEA, System for the Analysis of Global Energy Markets (2006).)

Other research needs with respect to photosynthetic hydrogen generation include:

• Detailed molecular investigations of the processes involved,
• Research into the possibility of switching biofilm systems “on” and “off,”
• Research into the thermodynamic limitations of the system, including the limits on photosynthetic biomass and hydrogen production,
• Research into improving the poor photosynthetic efficiency of phototrophic organisms,
• Research into the creation and utilization of possible co-products made by phototrophic bacteria, and
• Studies on the function of natural rhodopsin-based systems and their potential utility as a bio-based photosynthetic system for energy production.

METHANE

Methane is the end product of the anaerobic food chain—a sequence carried out by communities of fermentative bacteria, acid-producing intermediary organisms, and finally, methanogenic archaea. Methane-producing communities are very stable and resilient, but they are also complex and largely undefined. New species that engage in some aspect of methane production are frequently identified, but their functional and performance properties do not often differ substantially from that of known organisms.

Current commercial-scale methods of methane production yield from 50% to 100% conversion of substrate to methane on a mass basis, depending on the feedstock. Carbohydrate-rich substrates yield higher amounts of methane than feedstocks with high concentrations of lignocellulose or nitrogenous materials. The input feedstock also determines the composition of the gas product; carbohydrate substrates yield roughly 50% methane and 50% carbon dioxide, while more reduced (oxygen-poor) feedstocks yield higher proportions of methane.

Microbial Fuel Cells

A number of bacterial species are able to couple the oxidation of organic matter to the transfer of electrons to an electrode, thereby producing electricity. A microbial fuel cell is a system that makes use of this ability by feeding the microorganisms a constant supply of biomass and bypassing most of their biosynthetic processes in order to continuously generate electricity. Biomass substrates useful for fermentations can also be used in microbial fuel cells.

The arrangement of a microbial fuel cell is fairly simple: two chambers, one anaerobic and the other aerobic, are separated by a proton exchange membrane. Biomass fuel is fed to the bacteria on the anaerobic side. The bacteria then extract electrons from the biomass and deposit them on an anode that connects the two chambers, making a usable form of energy. The bacteria also extract protons from the biomass, allowing them to diffuse across the membrane to the cathode side. A catalyst on the aerobic cathode side combines the electrons, protons, and oxygen, making water. A primary goal of these systems is to minimize biosynthesis, which is a drain on the fuel cell reactants, in order to maximize electron flow.

The electrons that the bacteria extract from the substrate need a way to get to the electrode. They can either be shuttled to the electrode by chemical mediators like phenazine derivatives, or, if the strain is capable, directly from the cell through conductive appendages or through interconnected intercellular circuits.

The study of microbial fuel cells is in its infancy in comparison to other microbial energy technologies, and researchers still have a lot to learn about how to generate electrons. Efficiencies approaching 80% in shuttling electrons across the anode have been reported,⁴ but observations of greater than 95% efficiency are occurring.⁵ The most inefficient step of the process is the microbial interaction with the anode, on which the electrons from biomass are deposited. The rate of electron transfer to oxygen on the cathode side is also slow. The discovery of nanowires, electron conducting protein structures extending from the outer membrane of some bacterial cells, holds the promise for big improvements in anode performance.

Microbial fuel cells will likely be put to best use in situations in which energy needs are small but batteries are unsatisfactory. They may be used to power oceanographic instruments, for example, which are currently limited by battery power. Sediment fuel cells that exploit organic matter (or Fe(II), sulfide, or other electron source) and microbial communities in ocean sediments could potentially provide power for devices like these for many years.

A number of concepts hold promise for propelling microbial fuel cell technology forward. It may be possible to couple microbial redox systems with nanotechnology devices like nanoscale wires or nanotubes for electron transport or wires designed using biomimetics to imitate microbial pili, for example. Microbially produced electron shuttles, including phenazines, might also improve efficiency. Another possibility lies in combining phototrophs and chemoorganotrophs in multiple thin-layer photo-bioreactors. In such an arrangement, phototrophic microorganisms would make low molecular weight organic compounds to feed chemoorganotrophs continuously in the fuel cell.

Advantages

Microbial fuel cells have certain advantages over other microbial fuel technologies. They are reasonably efficient, and their ability to produce electricity continuously removes the constraint of seasonal variation in daylight intensity and photoperiod, factors that limit the productivity of hydrogen-generating photobioreactors. Microbial fuel cells are also adaptable to use in remote locations.

Disadvantages and Technical Difficulties

The disadvantages of microbial fuel cells is the slow rate that biomass is converted to electricity. Engineering advancements to increase the surface area of fuel cell electrodes is required because current scales directly with available surface area. Other practical difficulties include low current density, the scalability of the system, electrochemical resistances such as the rate of proton diffusion to the cathode, and increasing the rate of electron transfer between the microbes and the anode.

Research Needs

Microbial fuel cell research needs can be divided into microbio-logical issues and electrochemical issues. Crucial microbiological research topics include:

• Investigations into whether fuel cell efficiency can be improved through directed evolution of electricity-generating microorganisms. Bacteria have never before had any fitness incentive to generate electricity specifically, so applying selective pressure in the lab may well achieve success in improving fuel cell yield.
• Research into why some bacteria, such as Escherichia coli, can sustain continuous growth in a fuel cell while others, like Shewanella species, halt growth.
• Research into using other, new organisms as biocatalysts. The ability to transfer electrons to an anode is probably very broadly distributed in the bacterial world. Particular emphasis should focus on discovering:

  • Microbes with improved performance characteristics like longer nanowires to conduct electrons and better electron shuttling properties in general,
  • Thermophilic (heat-loving) and psychrophilic (cold-loving) organisms that can be used as biocatalysts, and
  • Diverse microbial communities that form functional biofilms on the anode in the presence of substrate that increase the efficiency of electron delivery.

Needed electrochemical research includes:

• Research into specific redox pairs that are amenable to fuel cell applications,
• Investigations into the electrochemical mechanisms that drive fuel cells. Currently, neither the mechanism of conveying electrons to the electrode nor the mechanism of electron conductance along nanowires are completely understood.
• Research into the regulation of electron transfer network synthesis and how it may be controlled or engineered into other microbes.

(Energy Information Administration/Annual Energy Outlook 2006)

BIODIESEL FUELS

It may be possible to use lipid-accumulating microorganisms like yeast or algae to produce oil for making biodiesel (a fuel made from transesterified vegetable oil). One advantage of this type of fuel production is the ease with which oil can be harvested from the system. However, emissions from biodiesel-powered vehicles are worse, in terms of their impacts on human and environmental health, than emissions from petrodiesel-powered vehicles. Hence, emission control equipment must be reconfigured in order to use biodiesel in a conventional diesel vehicle.

The possibility that high lipid-content microbes could be used as a resource for biodiesel production should be more fully explored. It is possible that the lipid content of these organisms could be manipulated to enhance yield.

BIOPROSPECTING

The scientific community needs a larger inventory of cultivated microorganisms from which to draw for microbial energy development. Bioprospecting can alleviate this shortfall. Also called “biodiversity prospecting,” bioprospecting seeks to catalog and capture the breadth of the world's biological diversity in order to put novel organisms and genes to work in energy production, industrial applications, medicine, or other areas.

Although metagenomics and new methods of identifying genes are important, and many of the functions desirable in energy production may be mined from metagenome samples (genomic DNA harvested from communities of microbes rather than single-species cultures), isolation of organisms with novel metabolisms and capabilities is still critical for the discovery of new and desirable metabolic functions. Hence, after carrying out metagenomic studies, the challenge becomes drawing out the microbes that encode the functions of interest from the environment. It is imperative to develop new methods for isolating microorganisms that have specific, desired functions.

The scientific community needs a larger inventory of cultivated microorganisms from which to draw for microbial energy development. Bioprospecting can alleviate this shortfall.

Careful design and implementation of multiple trait searching strategies that are guided by technical insights are recommended. This effort could be patterned after the successful efforts of the pharmaceutical industry that routinely carries out bioprospecting. The desired traits must be fully defined in an interdisciplinary effort to ensure that the isolates will meet energy conversion needs.

Biomining strategies that target microbes, microbial communities, and enzymes that can enhance the conversion of biomass to usable sugars should be implemented. The conversion of biomass to sugars is key to the creation of many end-product energy carriers, and lignocellulostic biomass is the most abundant resource of carbohydrates for these processes. However, despite the fact that this biomass is readily turned over in nature, few microbes that carry out the conversion of lignocellulose to sugars have been identified, suggesting that there is an untapped natural resource of microbes that can improve current technologies for the conversion of biomass into renewable fuels and chemicals. Metagenome sequencing of environments in which plant biomass is degraded at a high rate is recommended.

Advances are needed in our ability to cultivate heretofore unculturable microorganisms. It may be advisable to use a 96-well plate format for screening large numbers of organisms for desirable traits like electrical activity. In addition to finding new organisms, bioprospecting should also be used to isolate desirable traits that can then be engineered into “platform organisms,” those well studied that are amenable to manipulation.

(Energy Information Administration/Annual Energy Outlook 2006)

MICROBIAL COMMUNITIES

Many of the microbial processes critical to energy production are carried out by complex communities of organisms. There is a need to better understand the microbial community interactions that make these conversions possible. Better understanding of microbial community structure, robustness, networks, home-ostasis, and cell-to-cell signaling is also needed. A more thorough knowledge in all these areas will facilitate better modeling of microbial energy systems.

There is a need to study the fundamental mechanisms of energy distribution within microbial consortia, communities, and mats. One technique to do that is to apply high resolution techniques to determine the ATP contents of community members. Confocal microscopy, reporter genes, and adenylate charge ratio measurements would also be helpful in these studies.

Natural ecosystems containing complex microbial assemblages may contain organisms of potential interest in energy production. Studies of in situ metabolic activities and their regulation are warranted in order to optimize metabolic activities in vitro.

Tremendous advances have been made in imaging technologies that could allow a closer examination of biofilms and other complex communities to improve our control and use of these systems for energy transformation.

ENZYMOLOGY

A better understanding of the basic enzymology of microorganisms is necessary to move forward more quickly with microbial energy conversion. The actions of enzymes and enzyme complexes within the context of the whole cell, how they are regulated, where they are placed, what they interact with—all these topics need to be explored.

Nitrogenase is a good example of a poorly understood, but crucial, enzyme. There are still gaps in what is known about how the nitrogenase cofactor is assembled, why the enzyme needs ATP to function, which proteins are associated with the nitrogenase complex, and how the reductant gets carried to the enzyme. The enzymes of Photosystem II (PSII) also require further research. The exact placement of PSII in the cell membrane is critical to its function, and understanding how it is assembled and placed in the membrane could enable researchers to improve the system to direct the flow of light into energy-bearing products like hydrogen.

Research is needed to determine how best to apply metagenomic and directed evolution techniques to identify new, better enzymes for energy production and carbon sequestration. Annotation of putative gene products is distressingly poor. Improved approaches for determining the enzymatic function of predicted proteins would be most beneficial in the identification of alternative or improved metabolic pathways.

The bioinorganic chemistry of key catalytic enzymes should be studied in order to enable the design of bio-inspired synthetic catalysts. In addition, more knowledge of the enzymology of factors that limit potential efficient CO₂ removal (i.e., via RubisCO and other primary catalysts) is needed to help mitigate the effects of CO₂ production during cellulosic fermentations.

BIOLOGY OF NON-GROWING CELLS

One of the fundamental questions in microbial energy transformation is this: how can resting cells be induced to make energy products and not cell mass? In light of this challenge, there is a need to study and understand the behavior of these “metabolizing but non-growing” cells, including their maintenance energy requirements, the biology of starvation, the circumstances that bring about slow growth, and how to maintain non-growing cells at high densities. It is desirable to be able to turn growth-decoupled systems “on” and “off” rapidly.

MODELING AND MODEL ORGANISMS

Better modeling tools are needed to facilitate progress in microbial energy conversion. Models of metabolic dynamics, including concentrations of reductants and regulation of electron flow need to be improved. Experts in mechanistic and functional models should be included in model development in order to ensure that the metrics for each particular system are properly defined. Global techno-economic models of microbial energy transformation systems, which seek simultaneously to describe the resource flows into and out of a system as well as its economics, are needed and should be made publicly available on the internet.

Modeling methods that are user friendly for microbiologists and models that can enhance the dialog between chemical engineers and microbiologists are also needed.

It is important to continue the study of model organisms, as an understanding of model systems will advance not only the fundamental knowledge of microorganisms, but also the accuracy of genome and metagenome sequence annotation. It may be advisable to assemble a small collection of microorganisms that could serve as models for use in development of improved strains for energy conversion.

Broad investment in a range of strategies for microbial fuel production is recommended. It is in the interest of all parties to develop a diverse energy portfolio.

GENOMICS

The tools of genomics, proteomics, and metabolomics should be applied to determine how microbial cells carry out the functions of interest in microbial energy conversion, what limits these functions now, and what is likely to limit them in the future. It is also important to determine how these global processes are integrated with, and may be predicted from, genome sequences.

It is important to continue sequencing the complete genomes of microbes used in energy transformation, particularly since whole genome sequencing is now reasonably priced and the potential payoffs of sequencing are high.

The idea of using synthetic genomics to create entirely new organisms to carry out the desirable functions in microbial energy conversion has tremendous potential.

The metagenome sequences of targeted communities, including those found in the termite hindgut, rumen, microbial mats, and shipworm guts, could be highly useful in understanding and potentially manipulating biomass degradation for energy conversion.

NANOTECHNOLOGY

Nanotechnology could be a useful approach in designing bioreactors to improve product tolerance and in optically interrogating bioreactor membranes to evaluate stress. Micro- and nano-fluidics will eventually be useful for DNA fingerprinting linked with temporal and spatial analysis to examine system dynamics.

Nanotechnology tools, such as micromanipulation and fluidics, are likely to be useful in moving, capturing, and analyzing microbial cells in microbial energy systems. These capabilities would be useful in separating microbes with a DNA tag from the rest of the community, for example.

TECHNIQUES

The success of microbial energy conversion is largely dependent on the development of new and improved techniques for examining and optimizing microbial systems.

New and better methods are needed for measuring physiological processes on the microscale. Measuring the activities of single cells remains a technical challenge for microbiology. Singe cell measurements could enable insights into the stochasticity of microbial energy systems and the activity of unculturable microorganisms.

Improved ways of visualizing electron flow in real time are needed.

A better rubric for examining energy conversion efficiency is necessary to meaningfully compare different systems of microbial energy transformation and to evaluate their comparative advantages. For example, a system that currently operates at 0.1% conversion efficiency has more room for improvement and could merit more resource investment than a process that operates at 50% efficiency, but there is no widely accepted standard method for calculating efficiency in order to make such a comparison. The field also requires standardized methods for quantitatively evaluating the energy output of these systems.

Research in microbial energy conversion should make better use of the techniques of high-throughput technology in order to identify new microbes, enzymes, and products that can be put to use in energy transformation. High-throughput tools would also be useful for carrying out directed evolution of known enzymes in order to improve their catalytic efficiencies.

Rapid functional screens are needed to identify desirable microbial traits that can be used in energy conversion.

ADDITIONAL RESEARCH RECOMMENDATIONS

Other recommendations for moving forward with research in microbial energy conversions include:

• The development of pilot plants for microbial energy fuel production. Pilot plants are necessary but largely unavailable for small companies and universities to use for testing of their particular processes. The canyon between fundamental research and commercialization is massive and bridges are needed.
• Study the fundamentals of electron flow in microbes used in energy transformations. Electron flow is important for any organism that would produce electricity or a reduced chemical product. We also need an improved understanding of how different metabolic modules interact with each other for the production of energy fuels.
• Examine the communities of hydrogen-producing microbial mats. Microbial mats produce a tremendous amount of hydrogen, and we need an understanding of these communities and why they allow hydrogen to escape.
• Study microbial strategies that impact energy conversions but are not directly involved in the process. For example, we need a better understanding of the systems bacteria use to protect oxygen sensitive nitrogenases, hydrogenases, and methyl transferases.
• Apply systems biology in metabolic flux analysis to identify bottlenecks in energy-relevant microbial processes.
• Develop genetic exchange systems for new microbes to facilitate the construction of second-generation biocatalysts for the production of renewable chemicals and fuels.

EDUCATION AND TRAINING NEEDS

Research and development in microbial energy conversion call on the skills of individuals from multiple disciplines, not just microbiology. Educators must emphasize multidisciplinary training at both the undergraduate and graduate levels in order to meet the demands of this field of research. Some specific suggestions for improving the training of students destined for careers in microbial fuel production include:

• Requiring graduate students to complete rotations in different disciplines,
• Requiring internships or international study,
• Funding post-doctoral researchers who engage in multidisciplinary research,
• Funding sabbaticals for research professionals to study in a related field, and
• Developing university institutes in which researchers and students from many fields can have a common forum for discussing and sharing research in microbial energy conversion.

Educators must emphasize multidisciplinary training at both the undergraduate and graduate levels in order to meet the demands of this field of research.

Recent advances in genetic sequencing have made genomic data easy to generate, but analyzing those data remains a challenge. There is a need to train microbial physiologists who can interpret genomic data for use in the study of microbial energy conversion and elsewhere. University-level training in microbial physiology needs to be revamped and reenergized to meet this need. These training programs should include material that can facilitate collaborative efforts between physiologists and professionals in other disciplines (engineering, for example) to help solve the problems of energy transformation.

INTERDISCIPLINARY COOPERATION AND MULTIDISCIPLINARY RESEARCH

The study of microbial fuel production is multidisciplinary by nature: isolating and optimizing the microorganisms, designing and assembling the bioreactor or fuel cell, processing the biomass fuel, separating the products—these activities call on the skills of many different experts. Hence, strategies are needed to promote and advance cooperation between microbiologists, engineers, modelers, bioinformaticists, inorganic chemists, materials scientists, plant molecular biologists, plant breeders, and other experts involved in microbial energy research. Particular emphasis should be placed on assembling collaborations to make best use of genomics information and to develop innovative algorithms to apply to the specific needs of microbial energy conversion.

Unfortunately, mechanisms for funding joint research proposals in microbial energy are few and far between. It is important to create additional grant initiatives to fund these types of projects. Also, funding agencies often take a conservative approach in backing research by favoring projects that can muster a great deal of preliminary data. Relaxing the burden of proof required to secure funding would work to the advantage of fields like microbial energy conversion, which operate at the boundaries of the traditional departmental disciplines. Interdisciplinary training grants would also be helpful.

In carrying out multidisciplinary research, it is beneficial to assemble as many members of the research group as possible in close proximity, preferably within the same building. The development of core facilities is one mechanism for bringing scientists with diverse expertise together. One example of a successful interdisciplinary collaboration is the University of Southern California (USC) Interdisciplinary Microbial Fuel Cell group, which includes scientists from the USC departments of Earth Sciences (microbiology), Biology (molecular and evolutionary biology), Chemistry (membrane chemistry), and Engineering (modeling and electrochemistry) and houses them all in the same building. The group also has collaborations with scientists from other universities, including faculty from Penn State, Pacific Northwest National Labs, and the Korean Institute of Science and Technology.

THE ROLE OF PROFESSIONAL SOCIETIES IN MICROBIAL ENERGY RESEARCH

Professional societies should take the lead role in communicating research findings and opportunities in microbial fuel production in an accessible way to the public, legislators, and policy makers.

Professional societies must also take responsibility for educating their members about funding opportunities in microbial energy conversion available from private organizations.