Electricity generation in microbial fuel cells using neutral red as an electronophore

Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells.     

Extracellular Iron Reduction Is Mediated in Part by Neutral Red and Hydrogenase in Escherichia coli

Both microbial iron reduction and microbial reduction of anodes in fuel cells can occur by way of soluble electron mediators. To test whether neutral red (NR) mediates iron reduction, as it does anode reduction, by Escherichia coli, ferrous iron levels were monitored in anaerobic cultures grown with amorphous iron oxide. Ferrous iron levels were 19.4 times higher in cultures fermenting pyruvate in the presence of NR than in the absence of NR. NR did not stimulate iron reduction in cultures respiring with nitrate. To explore the mechanism of NR-mediated iron reduction, cell extracts of E. coli were used. Cell extract-NADH-NR mixtures had an enzymatic iron reduction rate almost 15-fold higher than the chemical NR-mediated iron reduction rate observed in controls with no cell extract. Hydrogen was consumed during stationary phase (in which iron reduction was detectable) especially in cultures containing both NR and iron oxide. An E. coli hypE mutant, with no hydrogenase activity, was also impaired in NR-mediated iron reduction activity. NR-mediated iron reduction rates by cell extracts were 1.5 to 2 times higher with hydrogen or formate as the electron source than with NADH. Our findings suggest that hydrogenase donates electrons to NR for extracellular iron reduction. This process appears to be analogous to those of iron reduction by bacteria that use soluble electron mediators (e.g., humic acids and 2,6-anthraquinone disulfonate) and of anode reduction by bacteria using soluble mediators (e.g., NR and thionin) in microbial fuel cells.     

Extracellular iron reduction is mediated in part by neutral red and hydrogenase in Escherichia coli

Both microbial iron reduction and microbial reduction of anodes in fuel cells can occur by way of soluble electron mediators. To test whether neutral red (NR) mediates iron reduction, as it does anode reduction, by Escherichia coli, ferrous iron levels were monitored in anaerobic cultures grown with amorphous iron oxide. Ferrous iron levels were 19.4 times higher in cultures fermenting pyruvate in the presence of NR than in the absence of NR. NR did not stimulate iron reduction in cultures respiring with nitrate. To explore the mechanism of NR-mediated iron reduction, cell extracts of E. coli were used. Cell extract-NADH-NR mixtures had an enzymatic iron reduction rate almost 15-fold higher than the chemical NR-mediated iron reduction rate observed in controls with no cell extract. Hydrogen was consumed during stationary phase (in which iron reduction was detectable) especially in cultures containing both NR and iron oxide. An E. coli hypE mutant, with no hydrogenase activity, was also impaired in NR-mediated iron reduction activity. NR-mediated iron reduction rates by cell extracts were 1.5 to 2 times higher with hydrogen or formate as the electron source than with NADH. Our findings suggest that hydrogenase donates electrons to NR for extracellular iron reduction. This process appears to be analogous to those of iron reduction by bacteria that use soluble electron mediators (e.g., humic acids and 2,6-anthraquinone disulfonate) and of anode reduction by bacteria using soluble mediators (e.g., NR and thionin) in microbial fuel cells.     

Improved fuel cell and electrode designs for producing electricity from microbial degradation

A new one-compartment fuel cell was composed of a rubber bunged bottle with a center-inserted anode and a window-mounted cathode containing an internal, proton-permeable porcelain layer. This fuel cell design was less expensive and more practical than the conventional two-compartment system, which requires aeration and a ferricyanide solution in the cathode compartment. Three new electrodes containing bound electron mediators including a Mn(4+)-graphite anode, a neutral red (NR) covalently linked woven graphite anode, and an Fe(3+)-graphite cathode were developed that greatly enhanced electrical energy production (i.e., microbial electron transfer) over conventional graphite electrodes. The potentials of these electrodes measured by cyclic voltametry at pH 7.0 were (in volts): +0.493 (Fe(3+)-graphite); +0.15 (Mn(4+)-graphite); and -0.53 (NR-woven graphite). The maximal electrical productivities obtained with sewage sludge as the biocatalyst and using a Mn(4+)-graphite anode and a Fe(3+)-graphite cathode were 14 mA current, 0.45 V potential, 1,750 mA/m(2) current density, and 788 mW/m(2) of power density. With Escherichia coli as the biocatalyst and using a Mn(4+)-graphite anode and a Fe(3+)-graphite cathode, the maximal electrical productivities obtained were 2.6 mA current, 0.28 V potential, 325 mA/m(2) current density, and 91 mW/m(2) of power density. These results show that the amount of electrical energy produced by microbial fuel cells can be increased 1,000-fold by incorporating electron mediators into graphite electrodes. These results also imply that sewage sludge may contain unique electrophilic microbes that transfer electrons more readily than E. coli and that microbial fuel cells using the new Mn(4+)-graphite anode and Fe(3+)-graphite cathode may have commercial utility for producing low amounts of electrical power needed in remote locations.     

Bioanode for a microbial fuel cell based on Gluconobacter oxydans immobilized into a polymer matrix

Acetic acid bacteria Gluconobacter oxydans subsp. industrius RKM V-1280 were immobilized into a synthetic matrix based on polyvinyl alcohol modified with N-vinylpyrrolidone and used as biocatalysts for the development of bioanodes for microbial fuel cells. The immobilization method did not significantly affect bacterial substrate specificity. Bioanodes based on immobilized bacteria functioned stably for 7 days. The maximum voltage (fuel cell signal) was reached when 100–130 μM of an electron transport mediator, 2,6-dichlorophenolindophenol, was added into the anode compartment. The fuel cell signals reached a maximum at a glucose concentration higher than 6 mM. The power output of the laboratory model of a fuel cell based on the developed bioanode reached 7 mW/m² with the use of fermentation industry wastes as fuel.     

Biocatalytic production of 5-hydroxy-2-adamantanone by P450cam coupled with NADH regeneration

5-Hydroxy-2-adamantanone is a versatile starting material for the synthesis of various adamantane derivatives. In this study, we investigated the biocatalytic production of 5-hydroxy-2-adamantanone using P450cam monooxygenase coupled with NADH regeneration. We constructed Escherichia coli cells that expressed P450cam and its redox partners, putidaredoxin and putidaredoxin reductase, and cells that co-expressed this P450cam multicomponent system with a glucose dehydrogenase (Gdh) to regenerate NADH using glucose. Two types of cells – wet cells that did not receive any treatment after washing with glycerol-containing buffer, and freeze-dried cells that were lyophilized after the washing – were prepared as whole-cell catalysts. When wet cells were reacted with 2-adamantanone, E. coli cells expressing only the P450cam multicomponent system efficiently produced 5-hydroxy-2-adamantanone in the presence of glucose. However, the co-expression of this P450cam system with Gdh did not further enhance the amount of this product. These results indicate that enough amounts of NADH for P450cam catalysis would be supplied by endogenous glucose metabolism in the E. coli host. In contrast, when freeze-dried cells were used, only the cells co-expressing the P450cam multicomponent system with Gdh efficiently catalyzed the oxidation in the presence of glucose. These results suggest that the exogenous Gdh compensated loss of NADH regeneration by the endogenous glucose metabolism that would be damaged by the lyophilization process. Furthermore, we attempted to produce 5-hydroxy-2-adamantanone with repeated additions of the substrate using wet cells expressing only the P450cam multicomponent system and freeze-dried cells co-expressing this P450cam system with Gdh. These whole-cell catalysts attained high-yield production; the wet cells and the freeze-dried cells produced 36 mM (5.9 g/l) and 21 mM (3.5 g/l) of 5-hydroxy-2-adamantanone, respectively.     

Microbial Utilization of Electrically Reduced Neutral Red as the Sole Electron Donor for Growth and Metabolite Production

Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H₂-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (−1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A. succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO₂. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed.     

Recovery of electric energy from formate by using a recombinant strain of Escherichia coli

Recombinant Escherichia coli cells were applied for the recovery of electric energy from formate. Initially, the fdh gene, which encodes formate dehydrogenase (FDH) of Mycobacterium vaccae, was introduced into E. coli cells to allow efficient degradation of formate. The constructed microbial fuel cell (MFC) with E. coli BW25113 cells carrying fdh gene showed appreciable generation of current density in the presence of formate as a substrate. Current density and polarization curves revealed that the performance of MFC under examined conditions was limited by the electron transfer from bulk liquid to the electrode surface; accordingly, agitation resulted in an increase in the current density and achieved a coulombic efficiency of 21.7 % on the basis of formate consumed. Thus, gene recombination enables E. coli cells to utilize formate as a fuel for MFC.     

Self-excreted mediator from <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 for electron transfer to carbon electrodes

Escherichia coli K-12 was cultured under anaerobic conditions to form biofilm on carbon fiber electrodes in glucose-containing medium. The anodic current increased with the development of the biofilm and depended on the glucose concentration. Cyclic voltammetric results support the presence of a redox compound(s) excreted from E. coli cells in the biofilm. The compound remained in the film under conditions of continuous flow and gave a couple of oxidation and reduction waves, which may be assigned to a menaquinone-like compound based on the mid-point potential (−0.22 V vs Ag|AgCl at pH 7.1) and its pH dependence. The catalytic current started to increase around the anodic peak potential of the redox compound and also increased by the permeabilization of the E. coli cell membranes with ethylenediamine tetraacetic acid-treatment. The results indicate that the E. coli-excreted redox compound works as a mediator for the electron transfer from the E. coli cells to the electrode as the final electron acceptor. The activity of the redox compound in the E. coli-biofilm as a mediator with some mobility was also verified for diaphorase-catalyzed electrochemical oxidation of NADH.     

Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells

This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pentose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric in vitro assays show no difference between the cofactors. Respiratory competent cells show cytosolic inhibition only when NADPH is produced, whereas production of NADH reveals uncoupling at low dicoumarol concentrations and inhibition of complexes III and IV at higher concentrations. Spectrophotometric assays only indicate the presence of cytosolic inhibition regardless of the reduced cofactor used. This article shows the applicability of the amperometric method and emphasizes the significance of determining biological effects of chemicals in living cells.     

Two dedicated class C radical S-adenosylmethionine methyltransferases concertedly catalyse the synthesis of 7,8-dimethylmenaquinone

Dimethylmenaquinone (DMMK), a prevalent menaquinone (MK) derivative of uncertain function, is characteristic for members of the class Coriobacteriia. Such bacteria are frequently present in intestinal microbiomes and comprise several pathogenic species. The coriobacterial model organism Adlercreutzia equolifaciens was used to investigate the enzymology of DMMK biosynthesis. A HemN-like class C radical S-adenosylmethionine methyltransferase (MenK2) from A. equolifaciens was produced in Wolinella succinogenes or Escherichia coli cells and found to methylate MK specifically at position C-7. In combination with a previously described MK methyltransferase (MqnK/MenK) dedicated to MK methylation at C-8, 7,8-DMMK₆ was produced in W. succinogenes. The position of the two methyl groups was confirmed by two-dimensional NMR and midpoint redox potentials of 7-MMK₆, 8-MMK₆ and 7,8-DMMK₆ were determined by cyclic voltammetry. A phylogenetic tree of MenK, MenK2 and HemN proteins revealed a Coriobacteriia-specific MenK2 clade. Using chimeric A. equolifaciens MenK/MenK2 proteins produced in E. coli it was shown that the combined linker and HemN domains determined the site-specificity of methylation. The results suggest that the use of MenK2 as a biomarker allows predicting the ability of DMMK synthesis in microbial species.     

Enhanced butanol production in a microbial electrolysis cell by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> IB4

Reducing power such as NADH is an essential factor for acetone/butanol/ethanol (ABE) fermentation using Clostridium spp. The objective of this study was to increase available NADH in Clostridium beijerinckii IB4 by a microbial electrolysis cell (MEC) with an electron carrier to enhance butanol production. First of all, a MEC was performed without electron carrier to study the function of cathodic potential applying. Then, various electron carriers were tested, and neutral red (NR)-amended cultures showed an increase of butanol concentration. Optimal NR concentration (0.1 mM) was used to add in a MEC. Electricity stimulated the cell growth obviously and dramatically diminished the fermentation time from 40 to 28 h. NR and electrically reduced NR improved the final butanol concentration and inhibited the acetone generation. In the MEC with NR, the butanol concentration, yield, proportion and productivity were increased by 12.2, 17.4, 7.2 and 60.3 %, respectively. To further understand the mechanisms of NR, cathodic potential applying and electrically reduced NR, NADH and NAD⁺ levels, ATP levels and hydrogen production were determined. NR and electrically reduced NR also improved ATP levels and the ratio of NADH/NAD⁺, whereas they decreased hydrogen production. Thus, the MEC is an efficient method for enhancing the butanol production.     

Lack of Electricity Production by Pelobacter carbinolicus Indicates that the Capacity for Fe(III) Oxide Reduction Does Not Necessarily Confer Electron Transfer Ability to Fuel Cell Anodes

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.     

Propionate Formation from Cellulose and Soluble Sugars by Combined Cultures of Bacteroides succinogenes and Selenomonas ruminantium

Succinate is formed as an intermediate but not as a normal end product of the bovine rumen fermentation. However, numerous rumen bacteria are present, e.g., Bacteroides succinogenes, which produce succinate as a major product of carbohydrate fermentation. Selenomonas ruminantium, another rumen species, produces propionate via the succinate or randomizing pathway. These two organisms were co-cultured to determine if S. ruminantium could decarboxylate succinate produced by B. succinogenes. When energy sources used competitively by both species, i.e. glucose or cellobiose, were employed, no succinate was found in combined cultures, although a significant amount was expected from the numbers of Bacteroides present. The propionate production per S. ruminantium was significantly greater in combined than in single S. ruminantium cultures, which indicated that S. ruminantium was decarboxylating the succinate produced by B. succinogenes. S. ruminantium, which does not use cellulose, grew on cellulose when co-cultured with B. succinogenes. Succinate, but not propionate, was produced from cellulose by B. succinogenes alone. Propionate, but no succinate, accumulated when the combined cultures were grown on cellulose. These interspecies interactions are models for the rumen ecosystem interactions involved in the production of succinate by one species and its decarboxylation to propionate by a second species.     

Oxygen exposure promotes fuel diversity for Shewanella oneidensis microbial fuel cells

Miniature microbial fuel cells (mini-MFCs) were used to monitor the current generated by Shewanella oneidensis DSP10 under both anaerobic and aerobic conditions when exposed to glucose as a potential electron donor. In addition to glucose, other carbon fuels including fructose, sucrose, acetate, and ascorbic acid were also tested. When the anolyte containing S. oneidensis was grown in the presence of oxygen, power densities of 270+/-10, 350+/-20, and 120+/-10 W/m(3) were recorded from the mini-MFC for glucose, fructose, and ascorbic acid electron donors, respectively, while sucrose and acetate produced no response. The power produced from glucose decreased considerably (相似文献   

The direct electrocatalysis of phenazine-1-carboxylic acid excreted by Pseudomonas alcaliphila under alkaline condition in microbial fuel cells

In this paper, we reported a kind of exoelectrogens, Pseudomonas alcaliphila (P. alcaliphila) strain MBR, which could excrete phenazine-1-carboxylic acid (PCA) to transfer electron under alkaline condition in microbial fuel cells (MFCs). The electrochemical activity of strain MBR and the extracellular electron transfer mechanism in MFCs were evaluated by cyclic voltammetry (CV) and electricity generation curve measurement. The results indicated a soluble mediator was the key factor for extracellular electron transfer of strain MBR under alkaline condition. The soluble mediator was PCA detected by gas chromatography-mass (GC-MS) analyses.     

Use of acetate for enrichment of electrochemically active microorganisms and their 16S rDNA analyses

A fuel cell-type electrochemical device has been used to enrich microbes oxidizing acetate with concomitant electricity generation without using an electron mediator from activated sludge. The device generated a stable current of around 5 mA with complete oxidation of 5 mM acetate at the hydraulic retention time of 2.5 h after 4 weeks of enrichment. Over 70% of electrons available from acetate oxidation was recovered as current. Carbon monoxide or hydrogen did not influence acetate oxidation or current generation from the microbial fuel cell (MFC). Denaturing gradient gel electrophoresis showed that DNA extracted from the acetate-enriched MFC had different 16S rDNA patterns from those of sludge or glucose+glutamate-enriched MFCs. Nearly complete 16S rDNA sequence analyses showed that diverse bacteria were enriched in the MFC fed with acetate. Electron microscopic observations showed biofilm developed on the electrode, but not microbial clumps observed in MFCs fed with complex fuel such as glucose and wastewater from a corn-processing factory.     

Reductive amination by recombinant Escherichia coli: Whole cell biotransformation of 2-keto-3-methylvalerate to l-isoleucine

A whole cell biotransformation system for reductive amination has been studied in recombinant Escherichia coli cells. Reductive amination of 2-keto-3-methylvalerate to l-isoleucine by a two-enzyme-cascade was achieved by overproduction of endogenous l-alanine dependent transaminase AvtA and heterologous l-alanine dehydrogenase from Bacillus subtilis in recombinant E. coli. Up to 100 mM l-isoleucine were produced from 100 mM 2-keto-3-methylvalerate and 100 mM ammonium sulfate. Regeneration of NADH as cofactor in the whole cell system was driven by glucose catabolism. The effects of defined gene deletions in the central carbon metabolism on biotransformation were tested. Strains lacking the NuoG subunit of NADH:ubiquinone oxidoreductase (complex I) or aceA encoding the glyoxylate cycle enzyme isocitrate lyase exhibited increased biotransformation rates.     

Biofuel Cells Select for Microbial Consortia That Self-Mediate Electron Transfer

Microbial fuel cells hold great promise as a sustainable biotechnological solution to future energy needs. Current efforts to improve the efficiency of such fuel cells are limited by the lack of knowledge about the microbial ecology of these systems. The purposes of this study were (i) to elucidate whether a bacterial community, either suspended or attached to an electrode, can evolve in a microbial fuel cell to bring about higher power output, and (ii) to identify species responsible for the electricity generation. Enrichment by repeated transfer of a bacterial consortium harvested from the anode compartment of a biofuel cell in which glucose was used increased the output from an initial level of 0.6 W m⁻² of electrode surface to a maximal level of 4.31 W m⁻² (664 mV, 30.9 mA) when plain graphite electrodes were used. This result was obtained with an average loading rate of 1 g of glucose liter⁻¹ day⁻¹ and corresponded to 81% efficiency for electron transfer from glucose to electricity. Cyclic voltammetry indicated that the enhanced microbial consortium had either membrane-bound or excreted redox components that were not initially detected in the community. Dominant species of the enhanced culture were identified by denaturing gradient gel electrophoresis and culturing. The community consisted mainly of facultative anaerobic bacteria, such as Alcaligenes faecalis and Enterococcus gallinarum, which are capable of hydrogen production. Pseudomonas aeruginosa and other Pseudomonas species were also isolated. For several isolates, electrochemical activity was mainly due to excreted redox mediators, and one of these mediators, pyocyanin produced by P. aeruginosa, could be characterized. Overall, the enrichment procedure, irrespective of whether only attached or suspended bacteria were examined, selected for organisms capable of mediating the electron transfer either by direct bacterial transfer or by excretion of redox components.     

Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.