The Low Biomass Yields of the Acetic Acid Bacterium Acetobacter pasteurianus Are Due to a Low Stoichiometry of Respiration-Coupled Proton Translocation

Growth energetics of the acetic acid bacterium Acetobacter pasteurianus were studied with aerobic, ethanol-limited chemostat cultures. In these cultures, production of acetate was negligible. Carbon limitation and energy limitation were also evident from the observation that biomass concentrations in the cultures were proportional to the concentration of ethanol in the reservoir media. Nevertheless, low concentrations of a few organic metabolites (glycolate, citrate, and mannitol) were detected in culture supernatants. From a series of chemostat cultures grown at different dilution rates, the maintenance energy requirements for ethanol and oxygen were estimated at 4.1 mmol of ethanol (middot) g of biomass(sup-1) (middot) h(sup-1) and 11.7 mmol of O(inf2) (middot) g of biomass(sup-1) (middot) h(sup-1), respectively. When biomass yields were corrected for these maintenance requirements, the Y(infmax) values on ethanol and oxygen were 13.1 g of biomass (middot) mol of ethanol(sup-1) and 5.6 g of biomass (middot) mol of O(inf2)(sup-1), respectively. These biomass yields are very low in comparison with those of other microorganisms grown under comparable conditions. To investigate whether the low growth efficiency of A. pasteurianus might be due to a low gain of metabolic energy from respiratory dissimilation, (symbl)H(sup+)/O stoichiometries were estimated during acetate oxidation by cell suspensions. These experiments indicated an (symbl)H(sup+)/O stoichiometry for acetate oxidation of 1.9 (plusmn) 0.1 mol of H(sup+)/mol of O. Theoretical calculations of growth energetics showed that this low (symbl)H(sup+)/O ratio adequately explained the low biomass yield of A. pasteurianus in ethanol-limited cultures.     

Glucose fermentation by Propionibacterium microaerophilum: effect of pH on metabolism and bioenergetic

pH affected significantly the growth and the glucose fermentation pattern of Propionibacterium microaerophilum. In neutral conditions (pH 6.5-7.5), growth and glucose fermentation rate (qs) were optimum producing propionate, acetate, CO(2), and formate [which together represented 90% (wt/wt) of the end products], and lactate representing only 10% (wt/wt) of the end products. In acidic conditions, propionate, acetate, and CO(2) represented nearly 100% (wt/wt) of the fermentation end products, whereas in alkaline conditions, a shift of glucose catabolism toward formate and lactate was observed, lactate representing 50% (wt/wt) of the fermentation end products. The energy cellular yields ( Y(X/ATP)), calculated (i) by taking into account extra ATP synthesized through the reduction of fumarate into succinate, was 6.1-7.2 g mol(-1). When this extra ATP was omitted, it was 11.9-13.1 g mol(-1). The comparison of these values with those of Y(X/ATP) in P. acidipropionici and other anaerobic bacteria suggested that P. microaerophilum could not synthesize ATP through the reduction of fumarate into succinate and therefore differed metabolically from P. acidipropionici.     

Biochemical characteristics and fermentation of glucose and starch by rabbit cæcal strains ofBifidobacterium globosum

Two strains ofBifidobacterium globosum were isolated from cæcal contents of rabbits in a search for potential probiotics. Both strains fermented glucose, galactose, pentoses, maltose, raffinose and starch. Common coccidiostats (monensin, salinomycin) and antimicrobial growth promotors (avoparcin, bacitracin, nitrovin, virginiamycin) supplied at 10 mg/L inhibited their growth in cultures with glucose. Fermentation parameters of bifidobacteria on glucose and starch. When growing on starch, the two strains of bifidobacteria produced 1 mol lactate per 5.6 and 5.7 mol acetate, respectively. Corresponding values during growth on glucose were 17.3 and 8.4 mol of acetate per mol of lactate. Starch-grown cells accumulated more saccharides than cells grown on glucose (1.48vs. 0.41 and 3.12vs. 1.18 mmol glucose units per 1 g of dry matter, respectively).     

The pH mediated effects of initial glucose concentration on the transitory occurrence of extracellular metabolites, gas exchange and growth yields of aerobic batch cultures of Klebsiella pneumoniae

The changes in growth kinetics in aerobic batch cultures of Klebsiella pneumoniae were followed by measurements of extracellular metabolites, rates of gas exchange, dissolved oxygen tension, pH, and carbon balance at all stages of growth. When the initial growth-limiting glucose concentration in media without pH control was increased from 1.0 g carbon L(-1) to 2.2 g carbon L(-1), the number of different, mainly acidic, extracellular metabolites of glucose at the end of exponential growth increased, while the proportion of acetate decreased. During the postexponential growth phase, the extracellular metabolites were oxidized, resulting in an increasing complexity of changes in pH, gas exchange, and dissolved oxygen tension with increasing initial substrate concentration. All these parameters showed concomitant stepwise changes. This pattern was independent of the dissolved oxygen tension in the range 30-200 muM. When pH was kept constant, the number, slope, and relative magnitude of the steps in gas exchange and dissolved oxygen tension were pH-dependent, being most complex at low pH. Detailed carbon balances showed that 20% of the initial glucose was converted into extracellular metabolites at the end of exponential growth at neutral pH. In the postexponential phase, pyruvate (2%) was reoxidized first followed by acetate (13%). The observed molar growth yield coefficient (Y(ATP)) was 8.4 if the transitory occurrence of pyruvate and acetate was accounted for, and 6.4 if it was neglected. The corrected observed molar growth yield coefficient (Y'(ATP)) was 9.4 and compared well with the true molar growth yield coefficient (Y(Max) (ATP)), which was found to be 11.0. Specific in situ respiration rates of the exponential growth phase of cultures grown at different controlled pH values compared well with in situ values for energy-limited chemostat grown cells at the same growth rates, suggesting that growth in the batch culture was energy-limited throughout the exponential growth phase. This view was supported by low levels of intracellular glycogen and exopolysaccharides of all cultures, by the value of Y'(ATP) of 9.4, and by a constant specific production rate of the extracellular metabolites throughout exponential growth. It was concluded that even under strictly aerobic conditions, control of pH is as important as control of dissolved oxygen tension during growth of enterobacteriaceae in batch cultures.     

Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Continuous Cultures

Rhodococcus erythropolis N1-36, a desulfurization strain, was grown in continuous culture at 10 different dilution rates with 50 (mu)M dibenzothiophene sulfone (DBTO(inf2)) as the growth-limiting nutrient. The steady-state biomass, concentrations of substrate (DBTO(inf2)) and product (monohydroxybiphenyl), saturation constant (0.39 (mu)M DBTO(inf2)), and cell yield coefficient (9 mg of biomass(middot)(mu)M(sup-1) DBTO(inf2)) were measured. Continuous cultures at five temperatures allowed calculation of activation energy (0.84 kcal(middot)mol(sup-1) [ca. 3.5 kJ(middot)mol(sup-1)]) near the optimal temperature (30(deg)C) for growth. A washout technique was used to calculate the maximum specific growth rate (0.235 h(sup-1)), a value equivalent to a minimum generation time of 2.95 h.     

Production of a Novel Extracellular Polysaccharide by Lactobacillus sake 0-1 and Characterization of the Polysaccharide

A novel exopolysaccharide (EPS) produced by Lactobacillus sake 0-1 (CBS 532.92) has been isolated and characterized. When the strain was grown on glucose, the produced EPS contained glucose and rhamnose in a molar ratio of 3:2 and the average molecular mass distribution (M(infm)) was determined at 6 x 10(sup6) Da. At a concentration of 1%, the 0-1 EPS had better viscosifying properties than xanthan gum when measured over a range of shear rates from 0 to 300 s(sup-1), while shear-thinning properties were comparable. Rheological data and anion-exchange chromatography suggested the presence of a negatively charged group in the EPS. Physiological parameters for optimal production of EPS were determined in batch fermentation experiments. Maximum EPS production was 1.40 g (middot) liter(sup-1), which was obtained when L. sake 0-1 had been grown anaerobically at 20(deg)C and pH 5.8. When cultured at lower temperatures, the EPS production per gram of biomass increased from 600 mg at 20(deg)C to 700 mg at 10(deg)C but the growth rate in the exponential phase decreased from 0.16 to 0.03 g (middot) liter(sup-1) (middot) h(sup-1). EPS production started in the early growth phase and stopped when the culture reached the stationary phase. Growing the 0-1 strain on different energy sources such as glucose, galactose, mannose, fructose, lactose, and sucrose did not alter the composition of the EPS produced.     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

Metabolism and Energetics of Lactococcus lactis during Growth in Complex or Synthetic Media

When Lactococcus lactis was grown in various complex or synthetic media, the fermentation of glucose remained homolactic whatever the medium used, with a global carbon balance of about 87%. Moreover, the nitrogen balance was not equilibrated, indicating that some amino acids led to the production of unknown nitrogen-containing carbon compounds while part of the glucose might contribute to anabolic pathways. In minimal medium containing six amino acids, a high concentration of serine was deaminated to pyruvate. This did not occur in more complete media, suggesting the presence of a regulation of this phenomenon by an amino acid. Ammonia produced during serine consumption was partly reconsumed after serine exhaustion. The values for biomass yield and biomass yield relative to ATP (Y(infATP)), the maximal growth rate, the specific rate of glucose consumption, and the corresponding rate of ATP synthesis all increased with the complexity of the medium, amino acid composition having the most pronounced effect. The Y(infATP) values were shown to range from 6.6 to 17.6 g of biomass(middot)mol of ATP(sup-1) on minimal and complex media.     

Xylose metabolism in Debaryomyces hansenii UFV-170. Effect of the specific oxygen uptake rate

The new yeast Debaryomyces hansenii UFV-170 was tested in this work in batch experiments under variable oxygenation conditions. To get additional information on its fermentative metabolism, a stoichiometric network was proposed and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. The yeast metabolism resulted to be practically inactive under strict oxygen-limited conditions (qO2 = 12.0 mmol(O2) C-mol(DM)(-1) h(-1)), as expected by the impossibility of regenerating NADH2+. Significant fractions of the carbon source were addressed to both respiration and biomass growth under excess oxygen levels (qO2 > or = 55.0 mmol(O2) C-mol(DM)(-1) h(-1)), thus affecting xylitol yield (Y(P/S) = 0.41-0.52 g g(-1)). Semi-aerobic conditions (qO2 = 26.8 mmol(O2) C-mol(DM)(-1) h(-1)) were able to ensure the best xylitol production performance (Pmax = 76.6 g L(-1)), minimizing the fractions of the carbon source addressed either to respiration or biomass production and increasing Y(P/S) up to 0.73 g g(-1). An average P/O ratio of about 1.0 mol(ATP) mol(O)(-1) allowed estimation of the main kinetic-bioenergetic parameters of the biosystem. The overall ATP requirements of biomass were found to be particularly high and dependent on the oxygen availability in the medium as well as on the physiological state of the culture. Under semi-aerobic and aerobic conditions, they varied in the ranges 13.5-15.4 and 9.74-10.2 mol(ATP) C-mol(DM)(-1), respectively, whereas during the best semi-aerobic bioconversion they progressively increased from 5.68 to 24.7 mol(ATP) C-mol(DM)(-1). After a starting phase of adaptation to the medium, the cell achieved a phase of decelerated growth during which its excellent xylose-to-xylitol capacity kept almost constant after 112 h up to the end of the run.     

Effects of Temperature, Water Activity, and Incubation Time on Production of Aflatoxins and Cyclopiazonic Acid by an Isolate of Aspergillus flavus in Surface Agar Culture

An experiment with a full factorial design was used to study the effects of and interactions among temperature, water activity (a(infw)), incubation period, and substrate on coproduction of aflatoxins (AF) and cyclopiazonic acid (CPA) by an isolate of Aspergillus flavus. Analysis of variance showed that there was a complex interaction among all of these factors and that this influenced the relative concentrations of the mycotoxins produced. The optimum temperatures for the production of AF and CPA were 30(deg)C and 25(deg)C, respectively. Both mycotoxins were maximally produced (0.306 to 0.330 (mu)g of AF(middot)ml of medium(sup-1), 4.040 to 6.256 (mu)g of CPA(middot)ml of medium(sup-1)) at an a(infw) of 0.996 and after 15 days of incubation. No AF were produced in either yeast extract agar or Czapek yeast autolysate agar medium at an a(infw) of 0.90 at 20 or 37(deg)C after 15 days (minimum conditions), while 0.077 to 0.439 (mu)g of CPA(middot)ml of medium(sup-1) was produced under the same conditions. Yeast extract agar favored maximum AF production, and Czapek yeast autolysate agar favored maximum CPA production.     

Fedbatch operation using Clostridium acetobutylicum suspension culture as biocatalyst for enhancing hydrogen production

We demonstrated the feasibility of fedbatch operation using Clostridium acetobutylicum suspension culture as a biocatalyst for the continuous production of hydrogen. The optimum operating pH and temperature of the current cultivation system for hydrogen production were pH 6.0 and 37 degrees C, respectively. The volumetric loading of the bioreactor for hydrogen production can be as high as 650 mmol hydrogen/L culture with a yield at approximately 2.0 mol hydrogen/mol glucose. Acetate and butyrate made up approximately 80% of the total metabolites. The inhibitory effect from the two metabolites on the hydrogen production process was investigated. Butyrate at a concentration higher than 13 g/L significantly inhibited not only cell growth but also hydrogen production (i.e., specific hydrogen production rate). Acetate appears to be less toxic than butyrate to the hydrogen production process. While significantly inhibiting cell growth, acetate hardly affected hydrogen production. Finally, the factors limiting cultivation performance were discussed and possible strategies for enhancing the production of hydrogen were proposed.     

一个新的高温产氢菌及产氢特性的研究

利用Hungate滚管技术从西藏山南地区热泉淤泥中分离到一株高温产氢的厌氧发酵细菌T42。菌株T42革兰氏染色反应为阴性,但KOH裂解试验证实其为革兰氏阳性杆菌。菌体大小为0.7μm~0.9μm×3.2μm~7μm,不运动,不产芽孢。其生长温度范围为32℃~69℃,最适生长温度为60℃~62℃,生长pH范围为5.0~8.8,最适生长pH为7.0~7.5,代时30min。有机氮源是T42菌株的必需生长因子。菌株T42利用淀粉、纤维二糖、蔗糖、麦芽糖、糊精、果糖、糖原和海藻糖等底物生长并发酵产氢,发酵葡萄糖的终产物为乙酸、乙醇、H2和CO2。G C含量为31.2mol%。系统发育分析表明菌株T42与Thermobrachium celere和Caloramator indicus位于同一分支,生理生化特征也表明菌株T42应是Thermobrachium属的一个新菌株,在中国普通微生物菌种保藏中心的保藏号为AS1.5039。菌株T42的最佳产氢初始pH为7.2,最佳产氢温度为62℃,其氢转化率为1.06mol H2/mol葡萄糖,最大产氢速率为24.0mmol H2/gDW/h。20mmol/L的Mg2 和2mmol/L的Fe2 可分别提高菌株T42的产氢量20%和23.3%,而Ni2 对其产氢无明显的作用。当菌株T42和热自养甲烷热杆菌(Methanothermobacter thermautotrophicus)Z245共培养时,由于降低了氢分压,使其葡萄糖利用率和氢产量分别提高1倍和2.8倍,发酵产物乙酸和乙醇的比例也从1提高到1.7。     

Effect of pH and stirring rate on itaconate production by Aspergillus terreus

The production of itaconic acid from glucose-based media by Aspergillus terreus NRRL 1960 was found to be controlled by stirring rate and pH. When the phosphorous (P) level in the production medium was reduced to less than 10 mg l(-1), the fungal mycelium exhausted its primary growth and started to excrete itaconic acid, while it continued its secondary growth at the expense of ammoniacal nitrogen. The fermentation exhibited a mixed-growth-associated product formation kinetics, the non-growth associated production term (mI) being practically zero only when the pH was left free to change from 3.4 down to 1.85. On the contrary, when the pH was kept reducing up to a constant value by automatic addition of KOH 4 mol l(-1), the itaconate yield coefficient on the initial glucose supplied (Y(I/So)) and mI and were 0.53 g g(-1) and 0.028 h(-1) at pH 2.4 and 320 rev min(-1) and 0.5 g g(-1) and 0.036 h(-1) at pH 2.8 and 400 rev min(-1), respectively. Although the differences between mI and Y(I/So) were statistically insignificant at the 95% confidence level, the net difference in the corresponding yield coefficients for itaconic acid on mycelial biomass resulted in a maximum itaconate production rate of 0.41 g l(-1) h(-1) at pH 2.8 and 400 rev min(-1), thus showing that this operating condition is no doubt optimal for the process under study.     

Effects of Low Dissolved-Oxygen Concentrations on Poly-(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Production by Alcaligenes eutrophus

The bacterial copolyester poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) was produced with Alcaligenes eutrophus DSM 545 from glucose and sodium propionate in a fed-batch fermentation with both nitrogen limitation and low dissolved-oxygen concentrations. When the dissolved-oxygen content was kept between 1 and 4% of air saturation during the polymer accumulation phase, the yield of 3-hydroxybutyrate (3HB) monomer from glucose was not affected, but the propionate-to-3-hydroxyvalerate (3HV) monomer yield was two to three times (0.48 to 0.73 mol of 3HV mol of propionate consumed(sup-1)) that observed in a control experiment (0.25 mol mol(sup-1)), where the accumulation-phase dissolved-oxygen concentration was 50 to 70% of air saturation. The overall polymer productivity of the fermentation was somewhat decreased by low dissolved-oxygen contents, owing to a slower 3HB production rate. The effect of a low dissolved-oxygen concentration is probably attributable to a reduction of the oxygen-requiring decarbonylation of propionyl-coenzyme A (CoA) to acetyl-CoA.     

Effect of organic complex compounds on Bacillus thermoamylovorans growth and glucose fermentation.

The effect of the concentration of a mixture (1/1 [wt/wt]) of yeast extract and bioTrypcase (YE+bT) on the growth and physiology of a new species, Bacillus thermoamylovorans, a moderately thermophilic, non-spore-forming, lactic acid-producing bacterium isolated from palm wine, was studied. At an initial glucose concentration of 100 mM, B. thermoamylovorans growth was limited when the concentration of YE+bT was lower than 5.0 g liter(-1); under these conditions, cellular yield reached a maximum value of 0.4 g of cells per g of YE+bT. Growth limitation due to deficiency in growth factors led to a significant shift in glucose metabolism towards lactate production. Lactate constituted 27.5 and 76% of the end products of glucose fermentation in media containing YE+bT at 20.0 and 1.0 g liter(-1), respectively. This result markedly differed from published data for lactic bacteria, which indicated that fermentative metabolism remained homolactic regardless of the concentration of YE. Our results showed that the ratio between cellular synthesis and energy production increased with the concentration of YE+bT in the culture medium. They indicate that the industrial production of lactic acid through glucose fermentation by B. thermoamylovorans can be optimized by using a medium where glucose is present in excess and the organic additives are limiting.     

Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.     

Acetoin production in growing Leuconostoc mesenteroides

Leuconostoc mesenteroides NCDO 518, provided with oxygen and pyruvate, preferentially used oxygen as accessory electron acceptor and converted pyruvate to acetoin. With glucose, 5.6 mM, as sole energy source only small amounts of acetoin were formed (0.08–0.21 mM). With glucose, 5.6 mM, and pyruvate, 20 mM, substantial amounts of acetoin were produced in growing, aerated cultures at pH 5 (2.8 mM, equivalent to 0.5 mol [mol glucose fermented]^–1). On exhaustion of glucose, growth ceased but metabolism of pyruvate continued with the formation of acetate and a little acetoin. In aerated cultures at pH 6 the general pattern was similar to that at pH 5 but less acetoin (0.6 mM) was formed during the growth phase and, after the exhaustion of glucose, pyruvate was converted very slowly to acetate only. Leuc. mesenteroides did not grow with pyruvate as sole energy source.     

Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate

In this study, local sewage sludge was acclimated to establish H2-producing enrichment cultures, which were used to convert sucrose to H2 with continuously stirred anaerobic bioreactors. The steady-state behaviors of cell growth, substrate utilization, and product formation were closely monitored. Kinetic models were developed to describe and predict the experimental results from the H2-producing cultures. Operation at dilution rates (D) of 0.075-0.167 h(-1) was preferable for H2 production, resulting in a H2 concentration of nearly 0.02 mol/l. The optimal hydrogen production rate was 0.105 mol/h occurring at D=0.125 h(-1). The major volatile fatty acid produced was butyric acid (HBu), while acetic acid and propionic acid were also produced in lesser quantities. The major solvent product was ethanol, whose concentration was only 15% of that of HBu, indicating that the metabolic flow favors H2 production. The proposed model was able to interpret the trends of the experimental data. The maximum specific growth rate (mu(max)), Monod constant (Ks), and yield coefficient for cell growth (Y(x/s)) were estimated as 0.172 h(-1), 68 mg COD/l, and 0.1 g/g, respectively. The model study also suggests that product formation in the continuous hydrogen-producing cultures was essentially a linear function of biomass concentration.     

The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

Azotobacter beijerinckii was grown in ammonia-free glucose-mineral salts media in batch culture and in chemostat cultures limited by the supply of glucose, oxygen or molecular nitrogen. In batch culture poly-beta-hydroxybutyrate was formed towards the end of exponential growth and accumulated to about 74% of the cell dry weight. In chemostat cultures little poly-beta-hydroxybutyrate accumulated in organisms that were nitrogen-limited, but when oxygen limited a much increased yield of cells per mol of glucose was observed, and the organisms contained up to 50% of their dry weight of poly-beta-hydroxybutyrate. In carbon-limited cultures (D, the dilution rate,=0.035-0.240h(-1)), the growth yield ranged from 13.1 to 19.8g/mol of glucose and the poly-beta-hydroxybutyrate content did not exceed 3.0% of the dry weight. In oxygen-limited cultures (D=0.049-0.252h(-1)) the growth yield ranged from 48.4 to 70.1g/mol of glucose and the poly-beta-hydroxybutyrate content was between 19.6 and 44.6% of dry weight. In nitrogen-limited cultures (D=0.053-0.255h(-1)) the growth yield ranged from 7.45 to 19.9g/mol of glucose and the poly-beta-hydroxybutyrate content was less than 1.5% of dry weight. The sudden imposition of oxygen limitation on a nitrogen-limited chemostat culture produced a rapid increase in poly-beta-hydroxybutyrate content and cell yield. Determinations on chemostat cultures revealed that during oxygen-limited steady states (D=0.1h(-1)) the oxygen uptake decreased to 100mul h(-1) per mg dry wt. compared with 675 for a glucose-limited culture (D=0.1h(-1)). Nitrogen-limited cultures had CO(2) production values in situ ranging from 660 to 1055mul h(-1) per mg dry wt. at growth rates of 0.053-0.234h(-1) and carbon-limited cultures exhibited a variation of CO(2) production between 185 and 1328mul h(-1) per mg dry wt. at growth rates between 0.035 and 0.240h(-1). These findings are discussed in relation to poly-beta-hydroxybutyrate formation, growth efficiency and growth yield during growth on glucose. We suggest that poly-beta-hydroxybutyrate is produced in response to oxygen limitation and represents not only a store of carbon and energy but also an electron sink into which excess of reducing power can be channelled.     

The Importance of Hydrogen in Landfill Fermentations

Forty-two samples taken from two landfills were monitored for CH(inf4) production and apparent steady-state H(inf2) concentration. The rates of methanogenesis in these samples ranged from below the detection limit to 1,900 (mu)mol kg (dry weight)(sup-1) day(sup-1), and the median steady-state hydrogen concentration was 1.4 (mu)M in one landfill and 5.2 (mu)M in the other. To further investigate the relationship between hydrogen concentration and methanogenesis, a subset of seven landfill samples was selected on basis of their rates of CH(inf4) production, H(inf2) concentrations, sample pHs, and moisture contents. Samples with H(inf2) concentrations of <20 nM had relatively small amounts of volatile fatty acids (VFAs) (undetectable to 18.6 mmol of VFA kg [dry weight](sup-1)), while samples with H(inf2) concentrations of >100 nM had relatively high VFA levels (133 to 389 mmol of VFA kg [dry weight](sup-1)). Samples with high H(inf2) and VFA contents had relatively low pH values (<=6.3). However, methanogenic and syntrophic bacteria were present in all samples, so the lack of methanogenesis in some samples was not due to a lack of suitable inocula. The low rates of methanogenesis in these samples were probably due to inhibitory effects of low pH and VFA accumulation, resulting from a thermodynamic uncoupling of fatty acid oxidation. As in other anaerobic ecosystems, H(inf2) is a critical intermediate that may be used to monitor the status of landfill fermentations.