Defeng Xing

Pyrosequencing reveals highly diverse microbial communities in microbial electrolysis cells involved in enhanced H2 production from waste activated sludge.

Renewable H(2) production from a plentiful biomass, waste activated sludge (WAS), can be achieved by fermentation, but the yields are low. The use of a microbial electrolysis cell (MEC) can increase the H(2) production yields to several times that of fermentation. We have proved that the enhancement of H(2) production was due to the ability of MECs to use a wider range of organic matter in WAS than in fermentation. To support this result strongly, we here investigated the microbial community structures of WAS and anode biofilms in WAS-fed MECs. A pyrosequencing analysis based on the bacterial 16S rRNA gene showed that dominant populations in MECs were more diverse than those in WAS (inoculum and substrate) after enrichment, and there was a clear distinction between MECs and WAS in microbial community structure. Diverse acid-producing bacteria and exoelectrogens (predominance of Geobacter) were detected in MECs but they were only rarely found in WAS. It has been reported that these acid-producing bacteria can ferment various sugars and amines with acetate, propionate, and butyrate as their major by-products. This was consistent with our chemical analyses. Detected exoelectrogens are known to use these organic acids (mainly acetate) and certain sugars to directly produce current for H(2) generation at the cathodes in the MECs. Using quantitative real-time PCR, we demonstrated that a consistent feed of alkaline-pretreated WAS containing large amounts of acetate led to a predominance of acetoclastic methanogens, while hydrogenotrophic methanogens were abundant in MECs fed both raw and alkaline-pretreated WAS. Syntrophic interactions between phylogenetically diverse microbial populations in anodophilic biofilms were found to drive the efficient cascade utilization of organic matter in WAS.

Isolation of the Exoelectrogenic Bacterium Ochrobactrum anthropi YZ-1 by Using a U-Tube Microbial Fuel Cell

ABSTRACT Exoelectrogenic bacteria have potential for many different biotechnology applications due to their ability to transfer electrons outside the cell to insoluble electron acceptors, such as metal oxides or the anodes of microbial fuel cells (MFCs). Very few exoelectrogens have been directly isolated from MFCs, and all of these organisms have been obtained by techniques that potentially restrict the diversity of exoelectrogenic bacteria. A special U-tube-shaped MFC was therefore developed to enrich exoelectrogenic bacteria with isolation based on dilution-to-extinction methods. Using this device, we obtained a pure culture identified as Ochrobactrum anthropi YZ-1 based on 16S rRNA gene sequencing and physiological and biochemical characterization. Strain YZ-1 was unable to respire using hydrous Fe(III) oxide but produced 89 mW/m2 using acetate as the electron donor in the U-tube MFC. Strain YZ-1 produced current using a wide range of substrates, including acetate, lactate, propionate, butyrate, glucose, sucrose, cellobiose, glycerol, and ethanol. Like another exoelectrogenic bacterium (Pseudomonas aeruginosa), O. anthropi is an opportunistic pathogen, suggesting that electrogenesis should be explored as a characteristic that confers advantages to these types of pathogenic bacteria. Further applications of this new U-tube MFC system should provide a method for obtaining additional exoelectrogenic microorganisms that do not necessarily require metal oxides for cell respiration.

Simultaneous Cellulose Degradation and Electricity Production by Enterobacter cloacae in a Microbial Fuel Cell

ABSTRACT Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from many different biodegradable substrates. When cellulose is used as the substrate, electricity generation requires a microbial community with both cellulolytic and exoelectrogenic activities. Cellulose degradation with electricity production by a pure culture has not been previously demonstrated without addition of an exogenous mediator. Using a specially designed U-tube MFC, we enriched a consortium of exoelectrogenic bacteria capable of using cellulose as the sole electron donor. After 19 dilution-to-extinction serial transfers of the consortium, 16S rRNA gene-based community analysis using denaturing gradient gel electrophoresis and band sequencing revealed that the dominant bacterium was Enterobacter cloacae. An isolate designated E. cloacae FR from the enrichment was found to be 100% identical to E. cloacae ATCC 13047T based on a partial 16S rRNA sequence. In polarization tests using the U-tube MFC and cellulose as a substrate, strain FR produced 4.9 ± 0.01 mW/m2, compared to 5.4 ± 0.3 mW/m2 for strain ATCC 13047T. These results demonstrate for the first time that it is possible to generate electricity from cellulose using a single bacterial strain without exogenous mediators.

Hydrogen production with effluent from an ethanol–H2-coproducing fermentation reactor using a single-chamber microbial electrolysis cell

Hydrogen can be produced by bacterial fermentation of sugars, but substrate conversion to hydrogen is incomplete. Using a single-chamber microbial electrolysis cell (MEC), we show that additional hydrogen can be produced from the effluent of an ethanol-type dark-fermentation reactor. An overall hydrogen recovery of 83+/-4% was obtained using a buffered effluent (pH 6.7-7.0), with a hydrogen production rate of 1.41+/-0.08 m(3) H(2)/m(3) reactor/d, at an applied voltage of E(ap)=0.6 V. When the MEC was combined with the fermentation system, the overall hydrogen recovery was 96%, with a production rate of 2.11 m(3) H(2)/m(3)/d, corresponding to an electrical energy efficiency of 287%. High cathodic hydrogen recoveries (70+/-5% to 94+/-4%) were obtained at applied voltages of 0.5-0.8 V due to shorter cycle times, and repression of methanogen growth through exposure of the cathode to air after each cycle. Addition of a buffer to the fermentation effluent was critical to MEC performance as there was little hydrogen production using unbuffered effluent (0.0372 m(3) H(2)/m(3)/d at E(ap)=0.6 V, pH 4.5-4.6). These results demonstrate that hydrogen yields from fermentation can be substantially increased by using MECs.

Enhanced hydrogen production from waste activated sludge by cascade utilization of organic matter in microbial electrolysis cells

Fermentative hydrogen production from waste activated sludge (WAS) has low H2 yield because WAS contains limited amounts of carbohydrate suitable for use by hydrogen-producing bacteria. Here, augmentation of hydrogen production from WAS by microbial electrolysis cells (MECs) was implemented. H2 yields of 3.89±0.39 mg-H2/g-DS (5.67±0.61 mg-H2/g-VSS) from raw WAS and 6.78±0.94 mg-H2/g-DS (15.08±1.41 mg-H2/g-VSS) from alkaline-pretreated WAS were obtained in the two-chamber MECs (TMECs). This was several times higher than yields obtained previously by fermentation. Single-chamber MECs (SMECs) with low internal resistance showed a H2 production rate that 13 times that of TMECs with similar H2 yield when alkaline-pretreated WAS was used. However, methanogenesis was detected after several batch cycles. A yield balance calculation revealed that carbohydrates were not the only substrates for electrohydrogenesis. Protein and its acidification products, such as volatile fatty acids are also responsible for a portion of H2 generation in MEC. Characterization of WAS in TMECs by three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy with parallel factor analysis indicated that electrohydrogenesis reacted on the extracellular polymeric substances and intracellular substances of WAS. Cascade utilization of organic matter in MECs increased hydrogen production from WAS. MECs showed high hydrogen yield from WAS, fewer H2 sinks, and insensitivity to temperature. Optimizing MEC configurations and operation conditions and improving the pretreatment processes of WAS are necessary before practical application can take place on a large scale.

Genetic Diversity of Hydrogen-Producing Bacteria in an Acidophilic Ethanol-H2-Coproducing System, Analyzed Using the [Fe]-Hydrogenase Gene

ABSTRACT Hydrogen gas (H2) produced by bacterial fermentation of biomass can be a sustainable energy source. The ability to produce H2 gas during anaerobic fermentation was previously thought to be restricted to a few species within the genera Clostridium and Enterobacter. This work reports genomic evidence for the presence of novel H2-producing bacteria (HPB) in acidophilic ethanol-H2-coproducing communities that were enriched using molasses wastewater. The majority of the enriched dominant populations in the acidophilic ethanol-H2-coproducing system were affiliated with low-G+C-content gram-positive bacteria, Bacteroidetes, and Actinobacteria, based on the 16S rRNA gene. However, PCR primers designed to specifically target bacterial hydA yielded 17 unique hydA sequences whose amino acid sequences differed from those of known HPB. The putative ethanol-H2-coproducing bacteria comprised 11 novel phylotypes closely related to Ethanoligenens harbinense, Clostridium thermocellum, and Clostridium saccharoperbutylacetonicum. Furthermore, analysis of the alcohol dehydrogenase isoenzyme also pointed to an E. harbinense-like organism, which is known to have a high conversion rate of carbohydrate to H2 and ethanol. We also found six novel HPB that were associated with lactate-, propionate-, and butyrate-oxidizing bacteria in the acidophilic H2-producing sludge. Thus, the microbial ecology of mesophilic and acidophilic H2 fermentation involves many other bacteria in addition to Clostridium and Enterobacter.

Hydrogen production, methanogen inhibition and microbial community structures in psychrophilic single-chamber microbial electrolysis cells

Hydrogen production by fermentative bacteria and photosynthetic organisms is depressed under psychrophilic conditions. Here we describe single-chamber microbial electrolysis cells (MECs), which were enriched successfully at 4 °C or 9 °C and operated at these temperatures for the production of hydrogen. The rates of hydrogen production varied from 0.23 ± 0.03 to 0.53 ± 0.04 m3 H2 m−3d−1, and the hydrogen yield was in the range 2.66 ± 0.22 − 2.94 ± 0.02 mol H2 mol−1acetate. The energy efficiency based on electrical input ranged from 154 ± 9 to 200 ± 18% using an applied voltage of 0.6 V or 0.8 V; CH4 was not detected in any of the reactors. The separate MECs were first operated at 25 °C and methanogenesis was found to occur in them, however no CH4 was produced if the cells were operated at 4 °C or 9 °C. 16S rRNA gene clone libraries constructed for Archaea proved that the growth of hydrogenotrophic methanogens such as Methanobrevibacter arboriphilus cultured in MECs at 25 °C was effectively inhibited under psychrophilic conditions, and the growth of these organisms was initially suppressed in these MECs maintained at low temperatures. Bacterial 16S rRNA gene clone library analyses showed that temperature significantly influenced the community structure of the anode biofilms in MECs, and Geobacter psychrophilus was found to be the dominant psychrotolerant population in MECs maintained at 4 °C and 9 °C. Our study also demonstrated that frequent inoculation using pre-acclimatized suspensions of microbial fuel cells (MFCs) greatly facilitated the rapid formation of psychrophilic biofilms in MECs at low temperature.

Change in microbial communities in acetate- and glucose-fed microbial fuel cells in the presence of light.

Power densities produced by microbial fuel cells (MFCs) in natural systems are changed by exposure to light through the enrichment of photosynthetic microorganisms. When MFCs with brush anodes were exposed to light (4000 lx), power densities increased by 8-10% for glucose-fed reactors, and 34% for acetate-fed reactors. Denaturing gradient gel electrophoresis (DGGE) profiles based on the 16S rRNA gene showed that exposure to high light levels changed the microbial communities on the anodes. Based on 16S rRNA gene clone libraries of light-exposed systems the anode communities using glucose were also significantly different than those fed acetate. Dominant bacteria that are known exoelectrogens were identified in the anode biofilm, including a purple nonsulfur (PNS) photosynthetic bacterium, Rhodopseudomonas palustris, and a dissimilatory iron-reducing bacterium, Geobacter sulfurreducens. Pure culture tests confirmed that PNS photosynthetic bacteria increased power production when exposed to high light intensities (4000 lx). These results demonstrate that power production and community composition are affected by light conditions as well as electron donors in single-chamber air-cathode MFCs.

Electricity generation of single-chamber microbial fuel cells at low temperatures.

Practical applications of microbial fuel cells (MFCs) for wastewater treatment will require operation of these systems over a wide range of wastewater temperatures. MFCs at room or higher temperatures (20-35°C) are relatively well studied compared those at lower temperatures. MFC performance was examined here over a temperature range of 4-30°C in terms of startup time needed for reproducible power cycles, and performance. MFCs initially operated at 15°C or higher all attained a reproducible cycles of power generation, but the startup time to reach stable operation increased from 50 h at 30°C to 210 h at 15°C. At temperatures below 15°C, MFCs did not produce appreciable power even after one month of operation. If an MFC was first started up at temperature of 30°C, however, reproducible cycles of power generation could then be achieved at even the two lowest temperatures of 4°C and 10°C. Power production increased linearly with temperature at a rate of 33±4 mW °C(-1), from 425±2 mW m(-2) at 4°C to 1260±10 mW m(-2) at 30°C. Coulombic efficiency decreased by 45% over this same temperature range, or from CE=31% at 4°C to CE=17% at 30°C. These results demonstrate that MFCs can effectively be operated over a wide range of temperatures, but our findings have important implications for the startup of larger scale reactors where low wastewater temperatures could delay or prevent adequate startup of the system.

Syntrophic interactions drive the hydrogen production from glucose at low temperature in microbial electrolysis cells.

H(2) can be obtained from glucose by fermentation at mesophilic temperatures, but here we demonstrate that hydrogen can also be obtained from glucose at low temperatures using microbial electrolysis cells (MECs). H(2) was produced from glucose at 4°C in single-chamber MECs at a yield of about 6 mol H(2)mol(-1) glucose, and at rates of 0.25±0.03-0.37±0.04 m(3) H(2)m(-3)d(-1). Pyrosequencing of 16S rRNA gene and electrochemical analyses showed that syntrophic interactions combining glucose fermentation with the oxidization of fermentation products by exoelectrogens was the predominant pathway for current production at a low temperature other than direct glucose oxidization by exoelectrogens. Another syntrophic interaction, methanogenesis and homoacetogenesis, which have been found in 25°C reactors, were not detected in MECs at 4°C. These results demonstrate the feasibility of H(2) production from abundant biomass of carbohydrates at low temperature in MECs.