Prepared by Merry Buckley and Judy Wall.
The report details one of the world’s largest problems – the need for clean, renewable sources of energy.
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Imagine the future of energy. The future might look like a new power plant on the edge of town—an inconspicuous bioreactor that takes in yard waste and locally-grown crops like corn and wood chips and churns out electricity to area homes and businesses. Or the future may take the form of a stylish-looking car that refills its tank at hydrogen stations. Maybe the future of energy looks like a device on the roof of your own home – a small appliance, connected to the household electric system, that uses sunlight and water to produce the electricity that warms your home, cooks your food, powers your television, and washes your clothes.
All these futuristic energy technologies may become reality some day, thanks to the work of the smallest living creatures on earth: microorganisms. “Microbial energy conversion” is the shorthand term for technologies like these. In microbial energy technologies, microorganisms make fuels out of raw organic materials, thereby converting the chemical energy in the biomass into chemical energy in the form of ethanol or hydrogen, for example. In addition, microbes can convert solar energy to hydrogen. Those fuels are then burned to make electrical energy or, in the case of internal combustion engines, kinetic energy to power a car. Another technology that falls under the heading of microbial energy conversion is the microbial fuel cell, a bioreactor in which bacteria transform the chemical energy in biomass directly into electrical energy.
The world faces a potentially crippling energy crisis in the next 30 to 50 years. Global populations are climbing, driving an everincreasing demand for energy to power manufacturing, transportation, heat, and other needs. “World energy consumption is projected to increase by 71 percent from 2003 to 2030” (Energy Information Administration/International Energy Outlook 2006). Petroleum, the foundation of the current transportation system, peaked in production in the U.S. in the mid 1980s, and world production is projected to peak in the next 25 to 50 years. Moreover, the burning of fossil fuels and the resulting release of carbon dioxide and combustion pollutants has brought about global climate change, the effects of which we are only beginning to understand. The means of preventing the twin catastrophes of energy scarcity and environmental ruin is not clear, but one part of the solution may lie in microbial energy conversion.
The American Academy of Microbiology convened a colloquium March 10-12, 2006, in San Francisco, California, to discuss the production of energy fuels by microbial conversions. The status of research into various microbial energy technologies, the advantages and disadvantages of each of these approaches, research needs in the field, and education and training issues were examined, with the goal of identifying routes for producing biofuels that would both decrease the need for fossil fuels and reduce greenhouse gas emissions. Colloquium participants made a number of recommendations for moving forward with research and education in this field.
Microorganisms may be used to generate a number of fuels, including ethanol, hydrogen, methane, lipids, and butanol, among which ethanol production is the most mature technology. As a liquid, ethanol is relatively easy to store and it can fit into the existing fuel infrastructure. However, ethanol adsorbs water readily and cannot be shipped through common-carrier pipelines, which inevitably contain water. Processes that generate higher molecular weight alcohols, such as butanol, can be produced with similar technologies and are more compatible with existing infrastructure. Currently, the production of ethanol from the most abundant forms of biomass, namely cellulose and lignocellulose, is comparatively difficult and expensive. Future research in alcohol production needs to focus on increasing the productivity and yield of processes that make alcohol from biomass and processes that generate alternative alcohols.
Hydrogen, a potent energy carrier, may be produced in any of a variety of ways. Oxygenic photosynthesis in cyanobacteria and other microbes can be harnessed to make hydrogen from water in a promising technology that may meet energy needs in the long term. The resources needed for this process, water and sunlight, are in practically unlimited supply, but the efficiency of the process is low. We need new photobioreactor designs to help increase efficiency of light capture and hydrogen removal in these systems. Hydrogen also may be produced using the cellular machinery of nitrogen fixation, through fermentation of biomass, through iron metabolism in photosynthesis, by metabolizing carbon storage compounds, or by microbial mats.
Methane is another useful microbially-produced fuel. Methanogenesis is a relatively simple, predictable microbial process, and methane fits into the infrastructure in place of natural gas. Although methane production is a well-explored area, much of the current data have derived from operations optimized for waste disposal, not for methane generation. Research in methane production should focus on optimizing the productivity of methanogenic microbial communities and bioreactor design.
The study of microbial fuel cells is in its infancy, and yield and current density are low in today’s systems, but the potential to make great leaps of progress in yield and performance is great. Research in microbial fuel cells should focus, in part, on the discovery and development of novel bacteria capable of transferring electrons from biomass substrates to an electrode.
Overarching research needs in the field include bioprospecting, the search for novel microorganisms and genes that can aid in energy conversion. Research is also needed to explore the dynamics of microbial communities, enzymology, the biology of non-growing cells, modeling, genomics, nanotechnology, new microbiological techniques, and bioreactor engineering.
Specific education and training in microbial energy conversion technologies are needed to provide the foundation for insights that will allow major increases in biofuel generation. We need our brightest and best minds to meet the challenges of this multidisciplinary effort.
The microbial fuel cell (MFC) is a technology that converts carbon-rich waste into bioelectricity using the ability of certain microorganisms, called exoelectrogens, to transfer electrons outside the cell . As there has been an increase in the global demand for renewable energy, exploiting biomass—which are biopolymers such as cellulose, chitin and starch—seems to be a promising part of the solution for the future. It may be possible for MFCs to compete with other methods of producing electricity from biomass, as it directly produces electricity rather than requiring multiple steps. Extensive investigations in the last years have shown that MFCs can be used for production of bioelectricity from different substrates, from simple sugars to complex wastes, including various types of wastewater [2,3]. Advances in new electrode materials and reactor configurations have increased power densities to as much as 1.5 kW/m3 . As cellulose is the most abundant polysaccharide in nature, there have been several efforts undertaken to design efficient MFCs using cellulose as a fuel [5,6,7,8]. The prerequisite for cellulose-fed MFCs is the appropriate choice of microorganisms able to decompose cellulose and produce a current. However, cellulose is a very difficult substrate for microorganisms to use in an MFC due to its water insolubility and complex spatial structure. Until now only one bacterial strain has been found to show both cellulolytic and electrogenic activity [7,9].
Generally, turning cellulose into electricity needs a syntrophic consortium where communication between species leads to metabolic cooperation enabling decomposition of a complex substrate, like cellulose, into volatile acids that can be further used by exoelectrogens for current production . The type of substrate used in MFCs strongly affects bacterial consortium dynamics and influences the composition of a bacterial community and performance of the system [11,12]. One of the most important directions in development of MFC technologies, beside reactor design, is the investigation of improving current production by exoelectrogenic microorganisms. This is especially important for biomass-fed MFCs, where a consortium of bacteria are required to convert the biomass to chemical that can be used by the exoelectrogenic bacteria. These improvements may include efficient selection of strains from consortia, genetic engineering, or optimization of adapting conditions. For these reasons, the knowledge of bacterial communities that evolve in cellulose-fed MFCs may be crucial to enhancing the performance of MFCs fed with cellulosic substrates.
Previous studies on cellulose-fed MFCs have usually been conducted with two-chamber systems, where the power densities produced were generally low, and dissolved oxygen was completely excluded from the anolyte (Table S1, [13,14,15,16,17,18,19,20,21,22]). Rezaei et al.  used a bacterial consortium from paper recycling wastewater in a two-chamber MFC with carbon electrodes and ferricyanide catholyte. The maximum power density obtained in a cotton linter cellulose-fed MFC was 18 mW/m2. Cellulose from cotton linters was used as a substrate in another two-chamber MFC study with carbon paper electrodes, which was inoculated with sludge from a wastewater treatment plant . The maximum power density was 12 mW/m2 when dissolved oxygen was used as the final electron acceptor. Unfortunately, there was no investigation into the bacterial consortia that evolved in these two studies. Rismani-Yazdi et al.  used a bacteria consortium from cow rumen in a two-chamber MFC with graphite electrodes and ferricyanide catholyte and obtained 55 mW/m2 when the cellulose from cotton linters was a substrate. An analysis of the bacterial communities after operation in a MFC system revealed the community was dominated by Firmicutes and Betaproteobacteria. However, there was no information about the original inoculum, so changes in consortium dynamics were not shown. Ishii et al.  used an Avicel cellulose as a substrate in a two-chamber MFC inoculated with rice paddy field soil from which 10 mW/m2 power was obtained. They found a different bacterial consortium after operation, as Rhizobiales, Clostridiales and Chloroflexi were predominant. In another two-chamber MFC fed wheat straw hydrolysate, Bacteroidetes and Alpha-, Beta- and Deltaproteobacteria were found to be dominant in the community . Thus, there were no common bacterial communities identified with these complex substrates.
Single-chamber air-cathode MFCs can produce higher power densities than two-chamber MFCs with dissolved oxygen, due to the higher internal resistance of the two-chamber reactors. However, oxygen diffusion through the air cathode can adversely affect power production by anaerobic microorganisms, and the infusion of oxygen can alter bacterial communities compared to completely anaerobic systems. For example, only 2–10 mW/m2 was obtained for an air-cathode single-chamber MFC fed with corn stover, inoculated with domestic wastewater . The power production was increased to 475 mW/m2 when a more readily biodegradable substrate (diluted hydrolysate of corn stover) was used as the substrate as oxygen could be more effectively scavenged using this substrate . Unfortunately, no bacterial identification was made in those studies. Many other reports using air-cathode MFCs have used soluble and readily biodegradable fermentation end products, not particulate substrates, and the resulting consortia is almost always found to be dominated by Deltaproteobacteria. For example, Kiely et al.  reported that a microbial community developed in air-cathode MFCs fed with succinic acid was dominated by Geobacter sulfurreducens, while Geobacter sp. were also reported to dominate consortia where acetate was used as a substrate [24,25]. Geobacter sp. was also found to be the most abundant when MFCs were fed more complex substrates, such as potato wastewater .
Although a number of papers available in the literature regard the bacterial composition of the consortia operating in MFCs, there are only few reports on direct biocurrent production from cellulose, and still little is known about the community evolving in MFCs with air cathodes. Thus, the identification of cellulose-degrading and electrogenic microorganisms that develop as a result of MFC operation on cellulose substrate is lacking. The aim of this work was to investigate the bacteria genera in a consortium before and after operation in an air-cathode, cellulose-fed MFC, which brings new data about the consortium evolving in the presence of cellulosic substrate. The influence of a cellulose substrate on changes in the bacterial community was examined after stable power was generated in this system. The results of the investigations allow for a better understanding of the communities that develop in MFCs during bioelectricity production from complex substrates like cellulose.
2. Materials and Methods
2.1. MFC Construction and Operation
Single-chamber MFCs were constructed in duplicate as previously described by Logan et al. , based on a design used by many laboratories around the world . The cube-shaped MFC was made from Plexiglas by drilling a cylindrical chamber (4 cm long by 3 cm in diameter, 28 mL in volume). The anode was carbon fiber brush (2 cm long, 2.5 cm diameter) placed in the center of the chamber. The air cathode (7 cm2 area) was carbon cloth with four polytetrafluoroethylene (PTFE) diffusion layers, a Pt catalyst and the Nafion binder, as previously described . The consortium of anaerobic bacteria was isolated from cow manure collected from a farm near Warsaw, Poland. The cow manure was filtered and suspended in 50 mM phosphate buffer-saline (PBS) solution at a 25% concentration. Water insoluble cellulose fibers (Sigma C6288) from cotton linters, were used as the substrate (1%). The scheme of possible use of cotton linters and their residues after isolation of cellulose fibers is presented in Figure S1 .
Current and power generation in the MFC were determined by measuring the voltage (U) every 20 min with across fixed external resistance (1000 Ω, unless noted otherwise) with a self-made Arduino-based automated measuring system connected to a computer. Current (I) was calculated from Ohm’s law (I = U/R) and power (P) was calculated as P = IU. Current density and power density were normalized to the projected surface area of the cathode (7 cm2). In order to obtain the polarization curves using the single cycle method the external resistance was changed over a range 102–104 Ω, with 20 min per resistor.
2.2. DNA Extraction, Polymerase Chain Reaction (PCR) and Illumina Sequencing
Cow manure samples (fresh inoculum and 30 days after stable power production in MFCs) were frozen after collection and stored at −80 °C. For DNA extractions, the samples were thawed on ice and vortexed independently. DNA extraction, PCR and Illumina sequencing were made according to the protocol described by Liang et al. .
2.3. Determination of Volatile Acids Production
Samples were quantitatively analyzed for acetic acid content using static headspace gas chromatography. Analyses were performed on Agilent 7697A Headspace Sampler coupled to gas chromatograph Agilent 7890A with a flame-ionization detector and a split/splitless injector. Prior to analysis, 5 mL of sample was transferred into a 20 mL headspace vial. Headspace sampler conditions were as follows: oven temperature 50 °C, oven equilibration time 20 min, loop temperature 65 °C, temperature of the transfer line 70 °C, time of pressurization 0.2 min, time of loop fill 0.2 min, time of loop equilibration 0.05 min, injection time 0.7 min. gas chromatography (GC) conditions were as follows: injector—splitless (250 °C), detector: FID (250 °C), oven: initial temperature program was 60 °C. This temperature was held for 6 min and then increased 10 °C min−1 to 230 °C with a final isothermal period of 5 min; flow rate of carrier gas (helium) through the column was 1.3 mL min−1. Separation was performed on capillary column Stabilwax-DA (Restek, Bellefonte, PA, USA) with modified polyethylene glycol phase (30 m × 0.25 mm I.D. × 0.25 μm film thickness). Acetic acid was quantified with reference to a standard calibration curve. The same procedure was used to determine valeric, isovaleric, butyric and isobutyric acids. All analyses were performed in triplicate. The obtained data were analyzed using ChemStation software (Agilent Technologies, Santa Clara, CA, USA).
3.1. Performance of MFCs Fed with Cellulose and Production of Volatile Acids
After inoculation of the MFC, the voltage fluctuated between 0 and 50 mV for the first 12 days of operation (Figure 1a). After this, the voltage began to stabilize and slowly increase to reach a peak of 175 mV in the first fed-batch cycle. Solution replacement generated repeatable cycles that lasted approximately 6 days. These observations are in accordance with the previous reports that cellulose hydrolysis is the slowest step in degradation process . The measurements of volatile acids revealed a high concentration of acetic acid (Figure 1b). Acetate was the dominant intermediate and it reached a maximum of 220 mg/L after 7 days, and then decreased after 15 days of MFC operation. The concentration of other acids (valeric, isovaleric, butyric and isobutyric acids) was remarkably lower, and did not exceed 5 mg/L over the whole period of study (data not shown). In previous investigations acetate was also found to be the main intermediate produced during cellulose degradation, and its concentration was observed to decrease with electricity production [16,21].
Power density was measured after three repeatable voltage cycles (1 month of MFCs operation). Polarization data revealed the maximum current produced in the reactors was 331 mA/m2 (R = 100 Ω) and the maximum power production was 44 mW/m2 (R = 1000 Ω), (Figure 2). During the next 30 days of observation, the power density averaged 36 ± 10 mW/m2. This power density was low relative to that typically produced with acetate, but more similar to previous results when using insoluble cellulose as a substrate .
3.2. Microbial Community Analysis
The microbial communities were examined on the anode of the MFC, and compared to the inoculum. The main phylum that was enriched on the anode after 30 days of stable operation in MFC was Bacteroidetes, which increased from 0.8% in the inoculum to 34% in the MFC (Figure 3). Bacteroidetes have previously been reported to dominate the anode consortium of a two-chamber MFC fed wheat straw hydrolysate, and were also found in two-chamber acetate-fed MFCs [16,32] as well as in a single-chamber MFC . The most abundant phylum was Firmicutes, with 32% in the inoculum and 50% after 30 days. This observation is in accordance with a previous study where Firmicutes were found a dominating anodic communities of a cellulose-fed MFC . The relative abundance in two phyla: Proteobacteria and Actinobacteria remained at comparable levels in the inoculum and operation of the MFC. The presence of these two phyla was also observed in a two-chamber MFC fed cow manure .
At the genus level, in the Bacteroidetes phylum, the most abundant genera were Parabacteroides (relative abundance of 39%) and Proteiniphilum (33%) (Figure 4a). Amongha Firmicutes, the most abundant genera were Clostridium and Catonella (both with a relative abundance above 20%), (Figure 4b). Clostridium genus was previously found to be dominant in the community of a single-chamber MFC fed with lignocellulosic substrate , a single-chamber MFC fed with sucrose , and two-chamber MFC fed with acetate . The highest relative abundance of genera in the bacteria consortium evolved after working in the MFC system was observed for Parabacteroides (13%),