Effect of humic acids on electricity generation integrated with xylose degradation in microbial fuel cells

Pentose and humic acids (HA) are the main components of hydrolysates, the liquid fraction produced during thermohydrolysis of lignocellulosic material. Electricity generation integrated with xylose (typical pentose) degradation as well as the effect of HA on electricity production in microbial fuel cells (MFCs) was examined. Without HA addition the maximum power density increased from 39.5 mW/m(2) to 83 mW/m(2) when initial xylose concentrations increased from 1.5 to 30 mM, while coulombic efficiency ranged from 13.5% to 52.4% for xylose concentrations of 15 and 0.5 mM, respectively. Compared to controls where HAs were not added, addition of commercial HA resulted in increase of power density and coulombic efficiency, which ranged from 7.5% to 67.4% and 24% to 92.6%, respectively. Digested manure wastewater (DMW) was tested as potential mediator for power generation due to its content of natural HA, and although it could produce higher coulombic efficiency namely 32.2% than the control of 18.3%, showed lower power density which was approx. 57 mW/m(2) in comparison to power density of the control which was 69 mW/m(2). Presence of commercial HA or DMW in the anode chamber resulted in faster xylose degradation and formation of more oxidized products (acetate and formate) as well as less reduced products (lactate and ethanol) compared to the controls. The reduced power generation in the presence of DMW was attributed to the presence of bacterial inhibitors such as phenolic compounds. Therefore, new feedstocks for MFCs, containing both mediators and substrates, such as lignocellulose hydrolysates should be considered for their applicability in MFCs.     

Electricity production from xylose using a mediator-less microbial fuel cell

Electricity generation integrated with xylose degradation was investigated in a two-chamber mediator-less microbial fuel cell (MFC). Voltage output followed saturation kinetics as a function of xylose concentration for concentration below 9.7 mM, with a predicted maximum of 86 mV (6.3 mW m(-2) or 116 mW m(-3)) and half-saturation constant (K(s)) of 0.29 mM. Xylose concentrations from 0.5 mM to 1.5 mM resulted in coulombic efficiencies and maximum voltage ranging from 41+/-1.6% to 36+/-1.2% and 55+/-2.0 mV to 70+/-3.0 mV respectively. Xylose degradation rate increased with increasing xylose concentration up to 9.7 mM and the predicted maximum degradation rate was 0.13 mM h(-1) and K(s) of 3.0 mM. Stirring by nitrogen in the anode chamber led to 99+/-2.3 mV maximum voltage (8.4+/-0.4 mW m(-2) or 153+/-7.1 mW m(-3)) and 5.9+/-0.3% coulombic efficiency at MFC running time 180 h, which were respectively 17+/-1.2% and 37+/-1.8%, higher than those without stirring. The COD removal under stirring was 22.1+/-0.3%, which was slightly lower than that of 23.7+/-0.4% under no stirring. However, stirring resulted in 59% lower xylose degradation rate. This work demonstrates that xylose can be used in the MFC for electricity production. Comparatively higher electricity generation and coulombic efficiency can be obtained by adjusting initial xylose concentration and applying stirring in the anode chamber.     

Power generation from cellulose using mixed and pure cultures of cellulose-degrading bacteria in a microbial fuel cell

Microbial fuel cells (MFCs) have been used to generate electricity from various organic compounds such as acetate, glucose, and lactate. We demonstrate here that electricity can be produced in an MFC using cellulose as the electron donor source. Tests were conducted using two-chambered MFCs, the anode medium was inoculated with mixed or pure culture of cellulose-degrading bacteria Nocardiopsis sp. KNU (S strain) or Streptomyces enissocaesilis KNU (K strain), and the catholyte in the cathode compartment was 50mM ferricyanide as catholyte. The power density for the mixed culture was 0.188mW (188mW/m(2)) at a current of 0.5mA when 1g/L cellulose was used. However, the power density decreased as the cellulose concentration in the anode compartment decreased. The columbic efficiencies (CEs) ranged from 41.5 to 33.4%, corresponding to an initial cellulose concentration of 0.1-1.0g/L. For the pure culture, cellobioase enzyme was added to increase the conversion of cellulose to simple sugars, since electricity production is very low. The power densities for S and K strain pure cultures with cellobioase were 162mW/m(2) and 145mW/m(2), respectively. Cyclic voltammetry (CV) experiments showed the presence of peaks at 380, 500, and 720mV vs. Ag/AgCl for the mixed bacterial culture, indicating its electrochemical activity without an external mediator. Furthermore, this MFC system employs a unique microbial ecology in which both the electron donor (cellulose) and the electron acceptor (carbon paper) are insoluble.     

Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production

Clostridium tyrobutyricum produces butyrate, acetate, H(2), and CO(2) as its main fermentation products from glucose and xylose. To improve butyric acid and hydrogen production, integrational mutagenesis was used to create a metabolically engineered mutant with inactivated ack gene, encoding acetate kinase (AK) associated with the acetate formation pathway. A non-replicative plasmid containing the acetate kinase gene (ack) fragment was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome should inactivate the target ack gene and produce ack-deleted mutant, PAK-Em. Enzyme activity assays showed that the AK activity in PAK-Em decreased by approximately 50%; meanwhile, phosphotransacetylase (PTA) and hydrogenase activities each increased by approximately 40%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expression of protein with approximately 32 kDa molecular mass was reduced significantly in the mutant. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37 degrees C, with a lower specific growth rate of 0.14 h(-1) (vs 0.21 h(-1) for the wild type), likely due to the partially impaired PTA-AK pathway. However, the mutant produced 23.5% more butyrate (0.42 vs 0.34 g/g glucose) at a higher final concentration of 41.7 g/L (vs 19.98 g/L) as a result of its higher butyrate tolerance as indicated in the growth kinetics study using various intial concentrations of butyrate in the media. The mutant also produced 50% more hydrogen (0.024 g/g) from glucose than the wild type. Immobilized-cell fermentation of PAK-Em in a fibrous-bed bioreactor (FBB) further increased the final butyric acid concentration (50.1 g/L) and the butyrate yield (0.45 g/g glucose). Furthermore, in the FBB fermentation at pH 5.0 with xylose as the substrate, only butyric acid was produced by the mutant, whereas the wild type produced large amounts of acetate (0.43 g/g xylose) and lactate (0.61 g/g xylose) and little butyrate (0.05 g/g xylose), indicating a dramatic metabolic pathway shift caused by the ack deletion in the mutant.     

A comparison of air and hydrogen peroxide oxygenated microbial fuel cell reactors

In this study, a two-compartment continuous flow microbial fuel cell (MFC) reactor was used to compare the efficiencies of cathode oxygenation by air and by hydrogen peroxide. The MFC reactor had neither a proton-selective membrane nor an electron transfer mediator. At startup, the cathodic compartment was continuously aerated and the anodic compartment was fed with a glucose solution. An increase of electrical power generation from 0.008 to 7.2 mW m(-2) of anode surface with a steady-state potential of 215-225 mV was observed within a period of 12 days. The performance of the air-oxygenated MFC reactor progressively declined over time because of biofilm proliferation in the cathodic compartment. Oxygenation of the cathodic compartment using 300 mL d(-1) of 0.3% hydrogen peroxide solution resulted in a power density of up to 22 mW m(-2) (68.2 mA m(-2)) of anode surface at a potential of 340-350 mV. The use of H2O2 for oxygenation was found to improve the long-term stability of the MFC reactor.     

Effects of ptb knockout on butyric acid fermentation by Clostridium tyrobutyricum

Clostridium tyrobutyricum ATCC 25755 is an anaerobic, rod-shaped, gram-positive bacterium that produces butyrate, acetate, hydrogen, and carbon dioxide from various saccharides, including glucose and xylose. Phosphotransbutyrylase (PTB) is a key enzyme in the butyric acid synthesis pathway. In this work, effects of ptb knockout by homologous recombination on metabolic flux and product distribution were investigated. When compared with the wild type, the activities of PTB and butyrate kinase in ptb knockout mutant decreased 76 and 42%, respectively; meanwhile, phosphotransacetylase and acetate kinase increased 7 and 29%, respectively. However, ptb knockout did not significantly reduce butyric acid production from glucose or xylose in batch fermentations. Instead, it increased acetic acid and hydrogen production 33.3-53.8% and ≈ 11%, respectively. Thus, the ptb knockout did increase the carbon flux toward acetate synthesis, resulting in a significant decrease (28-35% reduction) in the butyrate/acetate ratio in ptb mutant fermentations. In addition, the mutant displayed a higher specific growth rate (0.20 h(-1) vs. 0.15 h(-1) on glucose and 0.14 h(-1) vs. 0.10 h(-1) on xylose) and tolerance to butyric acid. Consequently, batch fermentation with the mutant gave higher fermentation rate and productivities (26-48% increase for butyrate, 81-100% increase for acetate, and 38-46% increase for hydrogen). This mutant thus can be used more efficiently than the parental strain in fermentations to produce butyrate, acetate, and hydrogen from glucose and xylose.     

Anaerobic obligatory xylitol production in Escherichia coli strains devoid of native fermentation pathways

Anaerobic glucose oxidation was coupled to xylose reduction in a nonfermentative Escherichia coli strain expressing NADPH-dependent xylose reductase. Xylitol production serves as the primary means of NAD(P)(+) regeneration, as glucose is converted primarily to acetate and CO(2). The membrane-bound transhydrogenase PntAB is required to achieve the maximum theoretical yield of four moles of xylitol per mole of glucose consumed.     

Succinic acid production by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> from batch fermentation of mixed sugars

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8–26.8 g/L and a total yield of 0.59–0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14–0.20 g/g and formate 0.08–0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO₂ sparging could replace carbonate supply in the form of MgCO₃ without affecting the succinate yield.     

Effect of electron mediators on current generation and fermentation in a microbial fuel cell

Effects of select electron mediators [9,10-anthraquinone-2,6-disulfonic acid disodium salt (AQDS), safranine O, resazurin, methylene blue, and humic acids] on metabolic end-products and current production from cellulose digestion by Clostridium cellulolyticum in microbial fuel cells (MFCs) were studied using capillary electrophoresis and traditional electrochemical techniques. Addition of the mediator resazurin greatly enhanced current production but did not appear to alter the examined fermentation end-products compared to MFCs with no mediator. Assays for lactate, acetate, and ethanol indicate that the presence of safranine O, methylene blue, and humic acids alters metabolite production in the MFC: safranine O decreased the examined metabolites, methylene blue increased lactate formation, and humic acids increased the examined metabolites. Mediator standard redox potentials (E ₀) reported in the literature do not coincide with redox potentials in MFCs due presumably to the electrolytic complexity of media that supports bacterial survival and growth. Current production in MFCs: (1) can be effected by the mediator redox potential while in the media, which may be significantly shifted from E ₀, and (2) depended on the ability of the mediator to access the bacterial electron source, which may be cytoplasmic. In addition, some electron mediators had significant effects on metabolic end-products and therefore the metabolism of the organism itself. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

一株产电菌Nitratireductor sp. WJ5-4的筛选及产电分析

【目的】从生物垃圾燃料电池阳极淋洗液中分离一株产电菌WJ5-4,研究其产电特性。【方法】根据菌株的形态、生理生化性质及16S r RNA基因测序分析确定其种属,以该菌株为产电菌,以生物垃圾为底物,构建微生物燃料电池(Microbial fuel cell,MFC),研究在不同接种浓度和底物固含量条件下菌株的产电性能。【结果】菌株WJ5-4被初步鉴定属于Nitratireductor属,当接种量200 m L时可获得最大功率密度135.16 m W/m2、稳定电压370 m V和总有机碳(Total organic carbon,TOC)降解率41.46%。当底物固含量为23%时,可获得最大功率密度163.69 m W/m2、稳定电压434 m V和TOC降解率46.29%。【结论】WJ5-4菌能够利用较高固含量的生物垃圾产电,产电周期较长,为下一步微生物燃料电池处理生物垃圾提供科学依据。     

Metabolic analysis of acetate accumulation during xylose consumption by<Emphasis Type="Italic"> Paenibacillus polymyxa</Emphasis>

Paenibacillus polymyxa ATCC 12321 produced more acetic acid and less butanediol from xylose than from glucose. The product yields from xylose were ethanol (0.72 mol/mol sugar), (R,R)-2,3-butanediol (0.31 mol/mol sugar), and acetate (0.38 mol/mol sugar) while those from glucose were ethanol (0.74 mol/mol sugar), (R,R)-2,3-butanediol (0.46 mol/mol sugar), and acetate (0.05 mol/mol sugar). Higher acetate kinase activity and lower acetate uptake ability were found in xylose-grown cells than in glucose-grown cells. Furthermore, phosphoketolase activity was higher in xylose-grown cells than in glucose-grown cells. In fed-batch culture on xylose, glucose feeding raised the butanediol yield to 0.56 mol/mol sugar and reduced acetate accumulation to 0.04 mol/mol sugar.     

Electricity production from xylose in fed-batch and continuous-flow microbial fuel cells

A medium-scale (0.77 l) air-cathode, brush-anode microbial fuel cell (MFC) operated in fed-batch mode using xylose (20 mM) generated a maximum power density of 13 +/- 1 W/m(3) (673 +/- 43 mW/m(2)). Xylose was rapidly removed (83.5%) within 8 h of a 60-h cycle, with 42.1% of electrons in intermediates (8.5 +/- 0.2 mM acetate, 5.9 +/- 0.01 mM ethanol, 4.3 +/- 0.1 mM formate, and 1.3 +/- 0.03 mM propionate), 9.1% captured as electricity, 16.1% in the remaining xylose, and 32.7% lost to cell storage, biomass, and other processes. The final Coulombic efficiency was 50%. At a higher initial xylose concentration (54 mM), xylose was again rapidly removed (86.9% within 24 h of a 116-h cycle), intermediates increased in concentration (18.4 +/- 0.4 mM acetate, 7.8 +/- 0.4 mM ethanol and 2.1 +/- 0.2 mM propionate), but power was lower (5.2 +/- 0.4 W/m(3)). Power was increased by operating the reactor in continuous flow mode at a hydraulic retention time of 20 h (20 +/- 1 W/m(3)), with 66 +/- 1% chemical oxygen demand removal. These results demonstrate that electricity generation is sustained over a cycle primarily by stored substrate and intermediates formed by fermentation and that the intermediates produced vary with xylose loading.     

Biosynthesis of Poly-beta-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava

The potential of Pseudomonas pseudoflava to produce poly-beta-hydroxyalkanoates (PHAs) from pentoses was studied. This organism was able to use a hydrolysate from the hemicellulosic fraction of poplar wood as a carbon and energy source for its growth. However, in batch cultures, growth was inhibited completely at hydrolysate concentrations higher than 30% (vol/vol). When P. pseudoflava was grown on the major sugars present in hemicelluloses in batch cultures, poly-beta-hydroxybutyric acid (PHB) accumulated when glucose, xylose, or arabinose was the sole carbon source, with the final PHB content varying from 17% (wt/wt) of the biomass dry weight on arabinose to 22% (wt/wt) of the biomass dry weight on glucose and xylose. Specific growth rates were 0.58 h on glucose, 0.13 h on xylose, and 0.10 h on arabinose, while the specific PHB production rates based on total biomass ranged from 0.02 g g h on arabinose to 0.11 g g h on glucose. PHB weight-average molecular weights were 640,000 on arabinose and 1,100,000 on glucose and xylose. The absolute amount of PHB in the cells decreased markedly when nitrogen limitation was relaxed by feeding ammonium sulfate at the end of the PHB accumulation stage of the arabinose and xylose fermentations. Copolymers of beta-hydroxybutyric and beta-hydroxyvaleric acids were produced when propionic acid was added to shake flasks containing 10 g of glucose liter. The beta-hydroxyvaleric acid monomer content attained a maximum of 45 mol% when the initial propionic acid concentration was 2 g liter.     

Influence of the metabolism pathway on lactic acid production from hemicellulosic trimming vine shoots hydrolyzates using Lactobacillus pentosus

Hemicellulosic hydrolyzates from trimming wastes of vine shoots were proposed as a carbon source for lactic acid production by Lactobacillus pentosus CECT-4023T (ATCC-8041). These hydrolyzates are composed mainly of glucose (12.0 g/L), xylose (17.5 g/L) and arabinose (4.3 g/L). Acetic acid, the main subproduct, started to be produced after all of the glucose was completely depleted, showing that the acetic acid coproduction came only from the xylose and arabinose consumption. In the absence of glucose, the L. pentosus pathway shifts from homo to heterofermentative. Thus, L. pentosus can be considered a facultative heterofermentative organism, degrading hexoses (glucose) via the Embden-Meyerhoff-Parnas pathway and pentoses (xylose and arabinose) via the phosphoketolase pathway. Hydrolyzates were vacuum evaporated to increase the initial sugars concentration up to 35.4 g/L of glucose, 52.3 g/L of xylose, and 13.0 g/L of arabinose. Under these conditions the lactic acid concentration reached 46.0 g/L (Q(P) = 0.933 g/L.h, Y(P/S) = 0.78 g/g; Y(P/S) theoretical = 91.7%) and a clear product inhibition was observed. Additional experiments with synthetic sugars, in the absence of inhibitory compounds, indicate that this inhibition must be attributed to the metabolic pathway but not to the inhibitory compounds present in the fermentation broth.     

Bioconversion of waste office paper to L(+)-lactic acid by the filamentous fungus Rhizopus oryzae

L(+)-lactic acid production was investigated using an enzymatic hydrolysate of waste office automation (OA) paper in a culture of the filamentous fungus Rhizopus oryzae. In 4 d culture, 82.8 g/l glucose, 7 g/l xylose, and 3.4 g/l cellobiose contained in the hydrolysate were consumed to produce 49.1 g/l of lactic acid. The lactic acid yield and production rate were only 0.59 g/g and 16.3 g/l/d, respectively, only 75% and 61% of the results from the glucose medium. The low production rate from waste OA hydrolysate was elucidated by trials using xylose as the sole carbon source; in those trials, the lactic acid production rate was 7.3 g/l/d, only 28% that of glucose or cellobiose. The low lactic acid yield from waste OA hydrolysate was clarified by trials using artificial hydrolysates comprised of 7:2:1 or 7:1:2 ratios of glucose:cellobiose:xylose. For both, the lactic acid production rate of 17.4 g/l/d matched that of waste OA paper, while the lactic acid yield was similar to that of the glucose medium. This indicates that the production rate may be inhibited by xylose derived from hemicellulose, and the yield may be inhibited by unknown compounds derived from paper pulp.     

腐植酸肥料对生姜土壤微生物量和酶活性的影响

设4个处理:空白对照、等量腐植酸、等量无机养分和腐植酸复合肥.通过小区试验,研究了腐植酸肥料在生姜不同生育时期对土壤微生物量和3种重要酶活性的影响.结果表明:与不施肥处理比较,施用腐植酸使前期的微生物量碳增加、脲酶活性降低,后期的微生物量碳减少、脲酶活性提高;施用腐植酸增加土壤活跃微生物量、提高酸性磷酸酶活性、降低蔗糖酶活性,全生育期平均土壤活跃微生物量和酸性磷酸酶活性分别提高17.34%和11.40%、蔗糖酶活性降低10.57%.与施用等量无机养分处理比较,施用腐植酸复合肥也使前期的微生物量碳增加、脲酶活性降低,后期的微生物量碳减少、脲酶活性提高;施用腐植酸复合肥增加土壤活跃微生物量、提高酸性磷酸酶和蔗糖酶活性,全生育期平均土壤活跃微生物量、酸性磷酸酶和蔗糖酶活性分别提高18.61%、10.07%和7.61%.     

Growth kinetics of Chlorella vulgaris and its use as a cathodic half cell

The kinetics of growth of the algal species Chlorella vulgaris has been investigated using CO(2) as the growth substrate. The growth rate was found to increase as the dissolved CO(2) increased to 150 mg/L, but fell dramatically at higher concentrations. Increasing the radiant flux also increased growth rate. With a radiant flux of 32.3 mW falling directly on the 500 mL culture media, the growth rate reached up to 3.6 mg of cells/L-h. Both pH variation (5.5-7.0) and mass transfer rate of CO(2) (K(L)a between 6h(-1) and 17 h(-1)) had little effect on growth rate. Growing on glucose, the yeast Saccharomyces cerevisiae produced a stable 160 mV potential difference when acting as a microbial fuel cell anode with ferricyanide reduction at the cathode. The algal culture was observed to be a workable electron acceptor in a cathodic half cell. Using an optimum methylene blue mediator concentration, a net potential difference of 70 mV could be achieved between the growing C. vulgaris culture acting as a cathode and a 0.02 M potassium ferrocyanide anodic half cell. Surge current and power levels of 1.0 microA/mg of cell dry weight and 2.7 mW/m(2) of cathode surface area were measured between these two half cells.     

A re-assessment of bacterial growth efficiency: the heat production and membrane potential of Streptococcus bovis in batch and continuous culture

Glucose-limited, continuous cultures (dilution rate 0.1 h^-1) of Streptococcus bovis JB1 fermented glucose at a rate of 3.9 mol mg protein^-1 h^-1 and produced acctate, formate and ethanol. Based on a maximum ATP yield of 32 cells/mol ATP (Stouthamer 1973) and 3 ATP/glucose, the theoretical glucose consumption for growth would have been 2.1 mol mg protein^-1 h^-1. Because the maintenance energy requirement was 1.7 mol/mg protein/h (Russell and Baldwin 1979), virtually all of the glucose consumption could be explained by growth and maintenance and the Y_ATP was 30. Glucose-limited, continuous cultures produced heat at a rate of 0.29 mW/mg protein, and this value was similar to the enthalpy change of the fermentation (0.32 mW/mg protein). Batch cultures (specific growth rate 2.0 h^-1) fermented glucose at a rate of 81 mol mg protein^-1 h^-1, and produced only lactate. The heat production was in close agreement with the theoretical enthalpy change (1.72 versus 1.70 mW/mg protein), but only 80% of the glucose consumption could be accounted by growth and maintenance. The Y_ATP of the batch cultures was 25. Nitrogen-limited, glucose-excess, non-growing cultures fermented glucose at a rate of 6.9 mol mg protein^-1 h^-1, and virtually all of the enthalpy for this homolactic fermentation could be accounted as heat (0.17 mW/mg protein). The nitrogenlimited cultures had a membrane potential of 150 mV, and nearly all of the heat production could be explained by a futile cycle of protons through the cell membrane (watts = amperes x voltage where H⁺/ATP was 3). The membrane voltage of the nitrogen-limited cells was higher than the glucose-limited continuous cultures (150 versus 80 mV), and this difference in voltage explained why nitrogen-limited cultures consumed glucose faster than the maintenance rate. Batch cultures had a membrane potential of 100 mV, and this voltage could not account for increased glucose consumption (more than growth plus maintenance). It appears that another mechanism causes the increased heat production and lower growth efficiency of batch cultures.     

Kinetic and nuclear magnetic resonance studies of xylose metabolism by recombinant Zymomonas mobilis ZM4(pZB5)

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by (13)C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo (31)P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.     

Enhanced Propionate Formation by Propionibacterium freudenreichii subsp. freudenreichii in a Three-Electrode Amperometric Culture System

In order to influence the fermentation pattern of Propionibacterium freudenreichii towards enhanced propionate formation, growth and product formation with glucose and lactate as energy sources were studied in a three-electrode poised-potential amperometric culture system. With anthraquinone 2,6-disulfonic acid (E(0)' = -184 mV; poised electron potential = -224 mV) or cobalt sepulchrate (E(0)' = -350 mV; -390 mV) as mediator and an activated platinum working electrode, reduction of bacterially oxidized mediator occurred fast enough to keep more than 50% of the respective mediator (in minimum 0.4 mM) in the reduced state, up to a current of 2 mA. With glucose as substrate, 90.0 or 97.3% propionate was formed during exponential growth in the presence of 0.5 mM anthraquinone 2,6-disulfonic acid or 0.4 mM cobalt sepulchrate, respectively. Growth yields of 56.3 or 53.8 g of cell material per mol of substrate degraded were calculated, respectively, and the electrons were transferred quantitatively from the working electrode to the bacterial cells. With l-lactate, only 68.6 or 72.9% propionate was formed with the same mediators. The results are discussed with respect to energetics, electron transfer potentials, and potential application of the new technique in technical propionate production.