Deeper below the surface-transcriptional changes in selected genes of Clostridium beijerinckii in response to butanol shock.

The main bottleneck in the return of industrial butanol production from renewable feedstock through acetone-butanol-ethanol (ABE) fermentation by clostridia, such as Clostridium beijerinckii, is the low final butanol concentration. The problem is caused by the high toxicity of butanol to the production cells, and therefore, understanding the mechanisms by which clostridia react to butanol shock is of key importance. Detailed analyses of transcriptome data that were obtained after butanol shock and their comparison with data from standard ABE fermentation have resulted in new findings, while confirmed expected population responses. Although butanol shock resulted in upregulation of heat shock protein genes, their regulation is different than was assumed based on standard ABE fermentation transcriptome data. While glucose uptake, glycolysis, and acidogenesis genes were downregulated after butanol shock, solventogenesis genes were upregulated. Cyclopropanation of fatty acids and formation of plasmalogens seem to be significant processes involved in cell membrane stabilization in the presence of butanol. Surprisingly, one of the three identified Agr quorum-sensing system genes was upregulated. Upregulation of several putative butanol efflux pumps was described after butanol addition and a large putative polyketide gene cluster was found, the transcription of which seemed to depend on the concentration of butanol.

Transcriptome analysis of Clostridium beijerinckii adaptation mechanisms in response to ferulic acid.

Clostridium beijerinckii 4693:int with high ferulic acid (FA) tolerance was engineered and characterized in our lab. In this study, the minimum inhibition concentrations of FA against C. beijerinckii NCIMB 8052 (wild-type) and 4693:int were 1.0 and 1.5g/l, respectively; cell viability was 18.5% and 106.7%, respectively, in the presence of 0.5g/l FA. A comparative transcriptome analysis was carried out at two different growth stages to evaluate sensitivity to FA. Genes that were differentially expressed included those related to redox and associated cofactors, riboflavin metabolism, two-component system, glycolysis and butanoate metabolism, and DNA replication as well as those encoding ATP-binding cassette transporters. Cbei_2134 and Cbei_2135 encoding alkyl hydroperoxide reductases are thought to be involved in antibacterial and adaptation mechanisms in C. beijerinckii in the presence of FA.

Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling.

Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.

Butanol production by a Clostridium beijerinckii mutant with high ferulic acid tolerance.

A mutant strain of Clostridium beijerinckii, with high tolerance to ferulic acid, was generated using atmospheric pressure glow discharge and high-throughput screening of C. beijerinckii NCIMB 8052. The mutant strain M11 produced 7.24 g/L of butanol when grown in P2 medium containing 30 g/L of glucose and 0.5 g/L of ferulic acid, which is comparable to the production from non-ferulic acid cultures (8.11 g/L of butanol). When 0.8 g/L of ferulic acid was introduced into the P2 medium, C. beijerinckii M11 grew well and produced 4.91 g/L of butanol. Both cell growth and butanol production of C. beijerinckii M11 were seriously inhibited when 0.9 g/L of ferulic acid was added into the P2 medium. Furthermore, C. beijerinckii M11 could produce 6.13 g/L of butanol using non-detoxified hemicellulosic hydrolysate from diluted sulfuric acid-treated corn fiber (SAHHC) as the carbon source. These results demonstrate that C. beijerinckii M11 has a high ferulic acid tolerance and is able to use non-detoxified SAHHC for butanol production.

Optimized Transformation of Newly Constructed Escherichia coli-Clostridia Shuttle Vectors into Clostridium beijerinckii.

Three Escherichia coli-Clostridia shuttle vectors, pKBA411-MCS, pKBE411-MCS, and pKBM411-MCS, which contain p15A, ColE1, and pMB1 origins for replication in E. coli, respectively, along with the pAMB origin for replication in C. beijerinckii, were constructed and examined for their transformation efficiencies into Clostridium beijerinckii NCIMB8052. The transformation condition of pKBM411-MCS, which was optimized by varying resistance, buffer composition, and DNA concentration, was further employed for the transformation of the other plasmids, pKBA411-MCS and pKBE411-MCS into C. beijerinckii. It was found out that transformation efficiency is highly dependent on the origin of replication. The highest transformation efficiency of 7.44 × 10(3) colony-forming units per microgram of DNA was obtained at 5.0 kV cm(-1) field strength, 200 Ω resistance, 270 mM sucrose concentration, 150 ng µg(-1), and 3.0 µg DNA using pKBM411-MCS having pMB1 and pAMB origins of replication. The application of the newly constructed vector system was also investigated by introducing the putative alcohol dehydrogenase gene of C. beijerinckii.

Allopurinol-mediated lignocellulose-derived microbial inhibitor tolerance by Clostridium beijerinckii during acetone-butanol-ethanol (ABE) fermentation.

In addition to glucans, xylans, and arabinans, lignocellulosic biomass hydrolysates contain significant levels of nonsugar components that are toxic to the microbes that are typically used to convert biomass to biofuels and chemicals. To enhance the tolerance of acetone-butanol-ethanol (ABE)-generating Clostridium beijerinckii NCIMB 8052 to these lignocellulose-derived microbial inhibitory compounds (LDMICs; e.g., furfural), we have been examining different metabolic perturbation strategies to increase the cellular reductant pools and thereby facilitate detoxification of LDMICs. As part of these efforts, we evaluated the effect of allopurinol, an inhibitor of NAD(P)H-generating xanthine dehydrogenase (XDH), on C. beijerinckii grown in furfural-supplemented medium and found that it unexpectedly increased the rate of detoxification of furfural by 1.4-fold and promoted growth, butanol, and ABE production by 1.2-, 2.5-, and 2-fold, respectively. Since NAD(P)H/NAD(P)(+) levels in C. beijerinckii were largely unchanged upon allopurinol treatment, we postulated and validated a possible basis in DNA repair to account for the solventogenic gains with allopurinol. Following the observation that supplementation of allopurinol in the C. beijerinckii growth media mitigates the toxic effects of nalidixic acid, a DNA-damaging antibiotic, we found that allopurinol elicited 2.4- and 6.7-fold increase in the messenger RNA (mRNA) levels of xanthine and hypoxanthine phosphoribosyltransferases, key purine-salvage enzymes. Consistent with this finding, addition of inosine (a precursor of hypoxanthine) and xanthine led to 1.4- and 1.7-fold increase in butanol production in furfural-challenged cultures of C. beijerinckii. Taken together, our results provide a purine salvage-based rationale for the unanticipated effect of allopurinol in improving furfural tolerance of the ABE-fermenting C. beijerinckii.

Enhanced butanol production by immobilized Clostridium beijerinckii TISTR 1461 using zeolite 13X as a carrier.

Butanol production by cell immobilization onto porous materials-brick and zeolite 13X-was investigated using Clostridium beijerinckii TISTR 1461. Characterization results of two materials were completed to evaluate their potential as an immobilization carrier. Although zeolite has greater porosity than brick, it cannot be used for cell aggregation without treating with chemical. After immobilization, both materials can enhance butanol titers from 5.29 to 5.80g/L and 8.58g/L using brick and zeolite, respectively. Butanol to glucose yield also improved from 0.14 to 0.16g/g after immobilization. It was found that butanol production significantly increased due to an increase in buffering capacity, strong bonding between the zeolite surface and cell, and butanol tolerance. In addition, repeated batch fermentation was performed, demonstrating that cells immobilized onto zeolite 13X have high stability and potential for long-term use in continuous fermentation.

Acetone-butanol-ethanol (ABE) production by Clostridium beijerinckii from wheat straw hydrolysates: efficient use of penta and hexa carbohydrates.

ABE fermentation by Clostridium beijerinckii of steam-exploded and ozonated wheat straw hydrolysates was investigated. In steam-exploded hydrolysates, highest yields of 0.40 g/g ABE yield and 127.71 g ABE/kg wheat straw were achieved when the whole slurry from the pretreatment was used. In ozonated hydrolysates, 0.32 g/g ABE yield and 79.65 g ABE/kg wheat straw were obtained from washed ozonated wheat straw. Diverse effects were observed in steam explosion and ozonolysis of wheat straw which resulted in hemicellulose removal and acid insoluble lignin solubilization, respectively. SEM analysis showed structural differences in untreated and pretreated biomass. Depending on the operational strategy, after pretreatment and enzymatic hydrolysis, the glucose recovery ranged between 65.73-66.49% and 63.22-65.23% and the xylose recovery ranged between 45.19-61.00% and 34.54-40.91% in steam-exploded and ozonated hydrolysates, respectively. The effect of the main inhibitory compounds found in hydrolysates (oxalic acid, acetic acid, 5-hydroxymethylfurfural and furfural) was studied through ABE fermentation in model media.

Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052.

Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon- and energy-limiting substrates, sucrose and the products of its hydrolysis d-glucose and d-fructose and only d-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l(-1) of butanol and 26.3 g l(-1) of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g(-1), respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in d-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the d-fructose phosphotransferase transport system and delayed sporulation.

Butanol production from acid hydrolyzed corn fiber with Clostridium beijerinckii mutant.

Sulfuric acid treated corn fiber hydrolysate (SACFH) inhibited cell growth and the production of butanol (4.7±0.2 g/L) by Clostridium beijerinckii IB4 in P2 medium. Optimal medium components were determined using fractional factorial design. NH4HCO3, FeSO4·7H2O and CaCO3 were demonstrated to be significant components in the production of butanol. The Box-Behnken design and a corresponding quadratic model were used to predict medium components (NH4HCO3 1.96 g/L, FeSO4·7H2O 0.26 g/L and CaCO3 3.15 g/L) and butanol yield (9.5 g/L). The confirmation experiment, under the predicted optimal conditions, yielded a butanol level of 9.5±0.1g/L. This study indicates that the Box-Behnken design is an effective approach for screening the optimal medium components required for the production of butanol. It also demonstrates that SACFH, which has high levels of inhibitors such as furan and phenolic compounds, may be used as a renewable carbon source in the production of biofuels.

Butanol production from hemicellulosic hydrolysate of corn fiber by a Clostridium beijerinckii mutant with high inhibitor-tolerance.

A Clostridium beijerinckii mutant RT66 with considerable inhibitor-tolerance obtained by continuous culture was used for butanol production from non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). In fed-batch fermentation, 1.8L of diluted SAHHC containing 10 g/L of reducing sugar was provided during the acidogenic phase and 0.2L of concentrated SAHHC containing 300 g/L of reducing sugar was provided during the solventogenic phase. The mutant produced a total amount of solvents of 12.9 g/L, which consisted of 3.1 g/L of acetone, 9.3 g/L of butanol and 0.5 g/L of ethanol. A solvent yield of 0.35 g/g sugar and a productivity of 0.18 g/L h in 72 h were achieved. The remarkable inhibitor-tolerance of C. beijerinckii RT66 demonstrates that this may be an excellent strain for butanol production from ligocellulosic materials.

Use of proteomic analysis to elucidate the role of calcium in acetone-butanol-ethanol fermentation by Clostridium beijerinckii NCIMB 8052.

Calcium carbonate increases growth, substrate utilization, and acetone-butanol-ethanol (ABE) fermentation by Clostridium beijerinckii NCIMB 8052. Toward an understanding of the basis for these pleiotropic effects, we profiled changes in the C. beijerinckii NCIMB 8052 proteome that occur in response to the addition of CaCO(3). We observed increases in the levels of different heat shock proteins (GrpE and DnaK), sugar transporters, and proteins involved in DNA synthesis, repair, recombination, and replication. We also noted significant decreases in the levels of proteins involved in metabolism, nucleic acid stabilization, sporulation, oxidative and antibiotic stress responses, and signal transduction. We determined that CaCO(3) enhances ABE fermentation due to both its buffering effects and its ability to influence key cellular processes, such as sugar transport, butanol tolerance, and solventogenesis. Moreover, activity assays in vitro for select solventogenic enzymes revealed that part of the underpinning for the CaCO(3)-mediated increase in the level of ABE fermentation stems from the enhanced activity of these catalysts in the presence of Ca(2+). Collectively, these proteomic and biochemical studies provide new insights into the multifactorial basis for the stimulation of ABE fermentation and butanol tolerance in the presence of CaCO(3).

Anthrahydroquinone-2,6,-disulfonate (AH2QDS) increases hydrogen molar yield and xylose utilization in growing cultures of Clostridium beijerinckii.

H(2) production and xylose utilization were investigated using the fermentative culture Clostridium beijerinckii NCIMB 8052. Adding anthrahydroquinone-2,6-disulfonate (AH(2)QDS) increased the extent of xylose utilization by 56% and hydrogen molar yield by 24-37%. Enhanced hydrogen molar yield correlated with increased xylose utilization and increases in the acetate/butyrate product ratio. An electron balance indicated that AH(2)QDS shifted the electrons from the butyric acid pathway (NADH-dependent pathway) to the acetic acid pathway (non-NADH-dependent pathway), putatively creating a surplus of reducing equivalents that were then available for hydrogen production. These data demonstrate that hydrogen yield and xylose utilization can be manipulated by amending redox active molecules into growing cultures. This will impact biohydrogen/biofuel production by allowing physiological manipulations of growing cells for increased (or decreased) output of selected metabolites using amendments that are not consumed during the reactions. Although the current yield increases are small, they suggest a target for cellular alterations. In addition, increased xylose utilization will be critical to the fermentation of pretreated lignocellulosic feedstocks, which may have higher xylose content.

Optimization of butanol production from tropical maize stalk juice by fermentation with Clostridium beijerinckii NCIMB 8052.

Mixed sugars from tropical maize stalk juice were used to carry out butanol fermentation with Clostridium beijerinckii NCIMB 8052. Batch experiments employing central composite design (CCD) and response surface methodology (RSM) optimization were performed to evaluate effects of three factors, i.e. pH, initial total sugar concentration, and agitation rate on butanol production. Optimum conditions of pH 6.7, sugar concentration 42.2g/L and agitation rate 48 rpm were predicted, under which a maximum butanol yield of 0.27 g/g-sugar was estimated. Further experiments demonstrated that higher agitation facilitated acetone production, leading to lower butanol selectivity in total acetone-butanol-ethanol (ABE). While glucose and fructose are more preferable by C. beijerinckii, sucrose can also be easily degraded by the microorganism. This study indicated that RSM is a useful approach for optimizing operational conditions for butanol production, and demonstrated that tropical maize, with high yield of biomass and stalk sugars, is a promising biofuel crop.

Outgrowth inhibition of Clostridium beijerinckii spores by a bacteriocin-producing lactic culture in ovine milk cheese.

In the manufacture of model cheeses, ovine milk was deliberately contaminated with spores of Clostridium beijerinckii INIA 63, a wild isolate from Manchego cheese with late blowing defect, and inoculated with nisin- and lacticin 481-producing Lactococcus lactis subsp. lactis INIA 415 as starter, to test its potential to prevent the late blowing defect, or with L. lactis subsp. lactis INIA 415-2, a spontaneous mutant not producing bacteriocins. Cheeses made individually with the lactococcal strains, without clostridial spores, served as controls. Cheese made with clostridial spores and L. lactis subsp. lactis INIA 415-2 showed late blowing defect after 120days of ripening. Spoilt cheese also showed lower concentrations of lactic acid, and higher levels of acetic, propionic and butyric acids, and of other volatile compounds such as 2-propanol and 1-butanol, than control cheese. In addition, cheese made with the bacteriocin producer did not show any late blowing symptoms, despite its spore counts similar to those of blown cheese, pointing to outgrowth inhibition of C. beijerinckii spores by bacteriocins. Besides, cheese made with the bacteriocin producer showed similar concentrations of lactic acid and volatile compounds than control cheese. Inclusion of L. lactis subsp. lactis INIA 415 in starter cultures seems a feasible method to prevent late blowing defect in cheese without altering its sensory characteristics.

Meta-analysis and functional validation of nutritional requirements of solventogenic Clostridia growing under butanol stress conditions and coutilization of D-glucose and D-xylose.

Recent advances in systems biology, omics, and computational studies allow us to carry out data mining for improving biofuel production bioprocesses. Of particular interest are bioprocesses that center on microbial capabilities to biotransform both the hexose and pentose fractions present in crop residues. This called for a systematic exploration of the components of the media to obtain higher-density cultures and more-productive fermentation operations than are currently found. By using a meta-analysis approach of the transcriptional responses to butanol stress, we identified the nutritional requirements of solvent-tolerant strain Clostridium beijerinckii SA-1 (ATCC 35702). The nutritional requirements identified were later validated using the chemostat pulse-and-shift technique. C. beijerinckii SA-1 was cultivated in a two-stage single-feed-stream continuous production system to test the proposed validated medium formulation, and the coutilization of D-glucose and D-xylose was evaluated by taking advantage of the well-known ability of solventogenic clostridia to utilize a large variety of carbon sources such as mono-, oligo-, and polysaccharides containing pentose and hexose sugars. Our results indicated that C. beijerinckii SA-1 was able to coferment hexose/pentose sugar mixtures in the absence of a glucose repression effect. In addition, our analysis suggests that the solvent and acid resistance mechanisms found in this strain are differentially regulated compared to strain NRRL B-527 and are outlined as the basis of the analysis toward optimizing butanol production.

Hydrogen production by the newly isolated Clostridium beijerinckii RZF-1108.

A fermentative hydrogen-producing strain, RZF-1108, was isolated from a biohydrogen reactor, and identified as Clostridium beijerinckii on the basis of the 16S rRNA gene analysis and physiobiochemical characteristics. The effects of culture conditions on hydrogen production by C. beijerinckii RZF-1108 were investigated in batch cultures. The hydrogen production and growth of strain RZF-1108 were highly dependent on temperature, initial pH and substrate concentration. Yeast extract was a favorable nitrogen source for hydrogen production and growth of RZF-1108. Hydrogen production corresponded to cell biomass yield in different culture conditions. The maximum hydrogen evolution, yield and production rate of 2209ml H2/l medium, 1.97 mol H2/mol glucose and 104.20 ml H2/g CDWh(-1) were obtained at 9 g/l of glucose, initial pH of 7.0, inoculum volume of 8% and temperature of 35 °C, respectively. These results demonstrate that C. beijerinckii can efficiently produce H2, and is another model microorganism for biohydrogen investigations.

Bioproduction of butanol in bioreactors: new insights from simultaneous in situ butanol recovery to eliminate product toxicity.

Simultaneous acetone butanol ethanol (ABE) fermentation by Clostridium beijerinckii P260 and in situ product recovery was investigated using a vacuum process operated in two modes: continuous and intermittent. Integrated batch fermentations and ABE recovery were conducted at 37 °C using a 14-L bioreactor (7.0 L fermentation volume) containing initial substrate (glucose) concentration of 60 g/L. The bioreactor was connected in series with a condensation system and vacuum pump. Vacuum was applied continuously or intermittently with 1.5 h vacuum sessions separated by 4, 6, and 8 h intervals. A control ABE fermentation experiment was characterized by incomplete glucose utilization due to butanol toxicity to C. beijerinckii P260, while fermentation coupled with in situ recovery by both continuous and intermittent vacuum modes resulted in complete utilization of glucose, greater productivity, improved cell growth, and concentrated recovered ABE stream. These results demonstrate that vacuum technology can be applied to integrated ABE fermentation and recovery even though the boiling point of butanol is greater than that of water.

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from Clostridium beijerinckii.

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H(2)O(2) to each compound up to 0.5-1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 microM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64-74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.

Influence of reduced electron shuttling compounds on biological H2 production in the fermentative pure culture Clostridium beijerinckii.

Several reports suggest that extracellular electron shuttles influence fermentative metabolism in a beneficial manner for bioremediation and biotechnology strategies. The focus of this research was to characterize the effects of reduced electron shuttling molecules on fermentative H(2) production. Reduced electron shuttles may provide reducing equivalents to generate H(2), which influences alternate cellular processes. Electron shuttling compounds cycle between reduced-oxidized states and influence fermentative physiology. Clostridium beijerinckii fermentation was altered using a physiological approach that resulted in H(2) production with the reduced extracellular electron shuttle anthrahydroquinone-2,6,-disulfonate (AH(2)QDS) and biologically reduced humic substances as the primary electron donors. Cells were suspended in a buffer with an excess of the biological electron transfer molecule NAD(+), with AH(2)QDS (100-1000 microM) or biologically reduced humic substances (0.01-0.025 g/L) as the sole electron source. Increasing concentrations of AH(2)QDS and reduced humics increased H(2) production, while H(2) production was suppressed by Fe(III) hydroxides, which outcompeted the cells for electrons from the reduced shuttles, suggesting that the shuttles are in fact electron donors for H(2) production. Oxidized AQDS/humics did not increase H(2) production. Organic acid production shifted toward butyric acid in the presence of reduced electron shuttles, particularly with growing cells. Growth and hydrogen production rates in growing cells were initially faster in the presence of the reduced electron shuttles; however, the final biomass yield was inversely proportional to the starting AH(2)QDS concentration, which suggests that reduced shuttles may compete with anabolic cell processes for available energetic resources or that the shift to excess butyrate becomes toxic to the cells.