Kinetics of Denitrifying Growth by Fast-Growing Cowpea Rhizobia

Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO₃⁻, NO₂⁻, and N₂O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients (Y) and higher saturation constants (K_s) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: K_s, 3.4 and 3.8 μM; Y, 16.0 and 14.0 g · cells eq⁻¹; μ_max, 0.41 and 0.33 h⁻¹. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent K_s values were 41, 270, and 460 μM for nitrous oxide, nitrate, and nitrite, respectively; the K_s for A28 grown on nitrate was 250 μM. The results are kinetically and thermodynamically consistent in explaining why O₂ is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (μ_max, 0.41 h⁻¹) when A26 was grown with either O₂ or NO₃⁻ as an electron acceptor and were only slightly reduced when A28 was grown with NO₃⁻ (0.25 h⁻¹) as opposed to O₂ (0.33 h⁻¹).     

Kinetics and Metabolism of Cellulose Degradation at High Substrate Concentrations in Steady-State Continuous Cultures of Clostridium cellulolyticum on a Chemically Defined Medium

The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of Clostridium cellulolyticum with increasing amounts of carbon substrate. At a dilution rate (D) of 0.048 h⁻¹, biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose liter⁻¹ the cell density at steady state leveled off. The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose liter⁻¹, respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed. The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis. A kinetics study of C. cellulolyticum metabolism in cellulose saturation was performed by varying D with 18.1 g of cellulose liter⁻¹. Compared to cellulose limitation (M. Desvaux, E. Guedon, and H. Petitdemange, J. Bacteriol. 183:119–130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD⁺, and q_{NADH produced}/q_{NADH used} ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as D rose, and (iii) the maximum growth yield (Y) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield (Y) (19.4 g of biomass per mol of ATP) were lowered. C. cellulolyticum was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions. However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients. The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, Biotechnol. Bioeng. 67:327–335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells⁻¹ h⁻¹ could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells⁻¹ h⁻¹; (ii) while the NADH/NAD⁺ ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions. Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.     

Kinetics of Insoluble Cellulose Fermentation by Continuous Cultures of Ruminococcus albus

Data from analyses of continuous culture fermentation of insoluble cellulose by Ruminococcus albus 7 were used to derive constants for the rate of cellulose hydrolysis and fermentation, growth yield, and maintenance. Cellulose concentration was 1% in the nutrient reservoir, and hydraulic retention times of 0.5, 1.0, 1.5, 1.75, and 2.0 days were used. Concentrations of reducing sugars in the cultures were negligible (less than 1%) compared with the amount of hydrolyzed cellulose, indicating that cellulose hydrolysis was the rate-limiting step of the fermentation. The rate of utilization of cellulose depended on the steady-state concentration of cellulose and was first order with a rate constant (k) of 1.18 day⁻¹. The true microbial growth yield (Y) was 0.11 g g⁻¹, the maintenance coefficient (m) was 0.10 g g⁻¹ h⁻¹, and the maximum Y_ATP was 7.7 g of biomass (dry weight) mol of ATP⁻¹.     

Chemostat Enrichments of Human Feces with Resistant Starch Are Selective for Adherent Butyrate-Producing Clostridia at High Dilution Rates

Resistant starch (RS) enrichments were made using chemostats inoculated with human feces from two individuals at two dilution rates (D = 0.03 h⁻¹ and D = 0.30 h⁻¹) to select for slow- and fast-growing amylolytic communities. The fermentations were studied by analysis of short-chain fatty acids, amylase and α-glucosidase activities, and viable counts of the predominant culturable populations and the use of 16S rRNA-targeted oligonucleotide probes. Considerable butyrate was produced at D = 0.30 h⁻¹, which corresponded with reduced branched-chain fatty acid formation. At both dilution rates, high levels of extracellular amylase activity were produced, while α-glucosidase was predominantly cell associated. Bacteroides and bifidobacteria predominated at the low dilution rate, whereas saccharolytic clostridia became more important at D = 0.30 h⁻¹. Microscopic examination showed that within 48 h of inoculation, one particular bacterial morphotype predominated in RS enrichments at D = 0.30 h⁻¹. This organism attached apically to RS granules and formed rosette-like structures which, with glycocalyx formation, agglomerated to form biofilm networks in the planktonic phase. Attempts to isolate this bacterium in pure culture were repeatedly unsuccessful, although a single colony was eventually obtained. On the basis of its 16S rDNA sequence, this RS-degrading, butyrate-producing organism was identified as being a previously unidentified group I Clostridium sp. A 16S rRNA-targeted probe was designed using this sequence and used to assess the abundance of the population in the enrichments. At 240 h, its contributions to total rRNA in the chemostats were 5 and 23% at D = 0.03 and 0.30 h⁻¹, respectively. This study indicates that bacterial populations with significant metabolic potential can be overlooked using culture-based methodologies. This may provide a paradigm for explaining the discrepancy between the low numbers of butyrate-producing bacteria that are isolated from fecal samples and the actual production of butyrate.     

Inorganic Carbon Limitation and Chemical Composition of Two Freshwater Green Microalgae

Two freshwater chlorophytes, Chlorella vulgaris and Scenedesmus obliquus, were grown in inorganic carbon-limited continuous cultures in which HCO₃⁻ was the sole source of inorganic carbon. The response of the steady-state growth rate to the external total inorganic carbon concentration was reasonably well described by the Monod equation; however, the response to the internal nutrient concentration was only moderately well represented by the Droop equation when the internal carbon concentration was defined on a cellular basis. The Droop equation was totally inapplicable when total biomass (dry weight) was used to define internal carbon because the ratio of carbon to dry weight did not vary over the entire growth rate spectrum. In batch cultures, maximum growth rates were achieved at the CO₂ levels present in atmospheric air and at HCO₃⁻ concentrations of 3 mM. No growth was observed at 100% CO₂. Both nitrogen uptake and chlorophyll synthesis were tightly coupled to carbon assimilation, as indicated by the constant C/N and C/chlorophyll ratios found at all growth rates. The main influence of inorganic carbon limitation appears to be not on the chemical structure of the biomass, but rather on cell size; higher steady-state growth rates lead to bigger cells.     

Effect of Specific Growth Rate on Fermentative Capacity of Baker’s Yeast

The specific growth rate is a key control parameter in the industrial production of baker’s yeast. Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature. In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial Saccharomyces cerevisiae strain in aerobic, glucose-limited chemostat cultures was investigated. At specific growth rates (dilution rates, D) below 0.28 h⁻¹, glucose metabolism was fully respiratory. Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol · g of biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (D < 0.28 h⁻¹). This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.025 h⁻¹ to 20.5 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.28 h⁻¹. At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts. Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity. These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.     

Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Growth Kinetics of Attached Iron-Oxidizing Bacteria

A model of growth and substrate utilization for ferrous-iron-oxidizing bacteria attached to the disks of a rotating biological contactor was developed and tested. The model describes attached bacterial growth as a saturation function in which the rate of substrate utilization is determined by a maximum substrate oxidation rate constant (P), a half-saturation constant (K_s), and the concentration of substrate within the rotating biological contactor (S₁). The maximum oxidation rate constant was proportional to flow rate, and the substrate concentration in the reactor varied with influent substrate concentration (S₀). The model allowed the prediction of metabolic constants and included terms for both constant and growth-rate-dependent maintenance energies. Estimates for metabolic constants of the attached population of acidophilic, chemolithotrophic, iron-oxidizing bacteria limited by ferrous iron were: maximum specific growth rate (μ_max), 1.14 h⁻¹; half-saturation constant (K_s) for ferrous iron, 54.9 mg/liter; constant maintenance energy coefficient (m₁), 0.154 h⁻¹; growth-rate-dependent maintenance energy coefficient (m′), 0.07 h⁻¹; maximum yield (Y_g), 0.063 mg of organic nitrogen per mg of Fe(II) oxidized.     

In glucose-limited continuous culture the minimum substrate concentration for growth,smin, is crucial in the competition between the enterobacterium Escherichia coli and Chelatobacter heintzii,an environmentally abundant bacterium

The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters μ_max and K_s were determined for growth with glucose. For both strains, μ_max was determined in batch culture after different precultivation conditions. In the case of C. heintzii, μ_max was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h⁻¹ C. heintzii grew immediately with a μ_max of 0.17±0.03 h⁻¹. After five transfers in batch culture, μ_max had increased only slightly to 0.18±0.03 h⁻¹. A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D=0.075 h⁻¹ into glucose-excess batch medium E. coli grew only after an acceleration phase of ∼3.5 h with a μ_max of 0.52 h⁻¹. After 120 generations and several transfers into fresh medium, μ_max had increased to 0.80±0.03 h⁻¹. For long-term adapted chemostat-cultivated cells, a K_s for glucose of 15 μg l⁻¹ for C. heintzii, and of 35 μg l⁻¹ for E. coli, respectively, was determined in ¹⁴C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h⁻¹. Using the determined pure culture parameter values for K_s and μ_max, it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s_min). The values estimated for s_min were dependent on growth rate; at D=0.05 h⁻¹, it was 12.6 and 0 μg l⁻¹ for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h⁻¹, s_min for E. coli had to be raised to 34.9 μg l⁻¹ whereas s_min for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K_s value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s_min.     

Synergistic Interaction Between Anabaena and Zoogloea spp. in Carbon Dioxide-Limited Continuous Cultures

Flocs consisting of Anabaena and Zoogloea spp. were used as a model system for the study of planktonic phototroph-heterotroph interactions. In CO₂-limited continuous culture (3.2 μmol of NaHCO₃ liter⁻¹ h⁻¹, 1.5 μmol of glucose liter⁻¹ h⁻¹, pH 8.5, D = 0.026 h⁻¹), the biomass of the phototroph increased 8.6-fold due to association. However, direct CO₂ exchange accounted for only a 3.8-fold increase. When the glucose supply rate was increased to 7.5 μmol liter⁻¹ h⁻¹, there was a 26-fold increase in biomass. When CO₂ was supplied in excess, there was no difference due to association. In batch culture, using the same medium, the specific growth rate was 0.029 h⁻¹ for the phototroph alone and 0.047 h⁻¹ for the phototroph in association with the heterotroph. The stimulatory effect of the heterotroph was found only under CO₂-limiting conditions and was directly related to the concentration of organic matter supplied in the medium. Both the biomass and the growth rate of the Anabaena sp. were increased by association with the Zoogloea sp. Thus, dissolved organic matter may substitute for CO₂ to maximize both growth rate and biomass production by phototrophs when heterotrophic bacteria are present.     

Efficient Preparation of Enantiopure D-Phenylalanine through Asymmetric Resolution Using Immobilized Phenylalanine Ammonia-Lyase from Rhodotorula glutinis JN-1 in a Recirculating Packed-Bed Reactor

An efficient enzymatic process was developed to produce optically pure D-phenylalanine through asymmetric resolution of the racemic DL-phenylalanine using immobilized phenylalanine ammonia-lyase (RgPAL) from Rhodotorula glutinis JN-1. RgPAL was immobilized on a modified mesoporous silica support (MCM-41-NH-GA). The resulting MCM-41-NH-GA-RgPAL showed high activity and stability. The resolution efficiency using MCM-41-NH-GA-RgPAL in a recirculating packed-bed reactor (RPBR) was higher than that in a stirred-tank reactor. Under optimal operational conditions, the volumetric conversion rate of L-phenylalanine and the productivity of D-phenylalanine reached 96.7 mM h⁻¹ and 0.32 g L⁻¹ h⁻¹, respectively. The optical purity (ee _D) of D-phenylalanine exceeded 99%. The RPBR ran continuously for 16 batches, the conversion ratio did not decrease. The reactor was scaled up 25-fold, and the productivity of D-phenylalanine (ee _D>99%) in the scaled-up reactor reached 7.2 g L⁻¹ h⁻¹. These results suggest that the resolution process is an alternative method to produce highly pure D-phenylalanine.     

Growth Kinetics and Yield Coefficients of the Extreme Thermophile Thermothrix thiopara in Continuous Culture

Thermothrix thiopara did not appear to be stressed at high temperature (72°C). Both the actual and theoretical yields were higher than those of analogous mesophilic sulfur bacteria, and the specific growth rate (μ_max) was more rapid than that of most autotrophs. The specific growth rate (0.58 h⁻¹), specific maintenance rate (0.11 h⁻¹), actual molar growth yield at μ_max (Y_max = 16 g mol⁻¹), and theoretical molar growth yield (Y_G = 24 g mol⁻¹) were all higher for T. thiopara (72°C) than for mesophilic (25 to 30°C) Thiobacillus spp. The growth efficiencies for T. thiopara at 70 and 75°C (0.84 and 0.78) were significantly higher than at 65°C (0.47). Corresponding specific maintenance rates were highest at 65°C (0.41 h⁻¹) and lowest at 70 and 75°C (0.11 and 0.15 h⁻¹, respectively). Growth efficiencies of metabolically similar mesophiles were generally higher than for T. thiopara. However, the actual yields at μ_max were higher for T. thiopara because its theoretical yield was higher. Thus, at 70°C, T. thiopara was capable of deriving more metabolically useful energy from thiosulfate than were mesophilic sulfur bacteria at 25 and 30°C. The low growth efficiency of T. thiopara reflected higher maintenance expenditures. T. thiopara had higher maintenance rates than Thiobacillus ferroxidans or Thiobacillus denitrificans, but also attained higher molar growth yields. It is concluded that sulfur metabolism may be more efficient overall at extremely high temperatures due to increased theoretical yields despite increased maintenance requirements.     

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h⁻¹ and a maximum prodigiosin concentration of 80 ng of prodigiosin · mg of cell protein⁻¹ were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h⁻¹), and maximum prodigiosin concentrations were higher (1,500 ng · mg of protein⁻¹). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na⁺; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na⁺ up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH₂PO₄ above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion · liter⁻¹, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng · mg protein⁻¹) was observed in chemostat cultures at a dilution rate of 0.057 h⁻¹. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h⁻¹, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.     

Aerobic Fermentation of D-Xylose to Ethanol by Clavispora sp

Eleven strains of an undescribed species of Clavispora fermented D-xylose directly to ethanol under aerobic conditions. Strain UWO(PS)83-877-1 was grown in a medium containing 2% D-xylose and 0.5% yeast extract, and the following results were obtained: ethanol yield coefficient (ethanol/D-xylose), 0.29 g g⁻¹ (57.4% of theoretical); cell yield coefficient (dry biomass/D-xylose), 0.25 g g⁻¹; maximum ethanol concentration, 5.9 g liter⁻¹; maximum volumetric ethanol productivity, 0.11 g liter⁻¹ h⁻¹. With initial D-xylose concentrations of 40, 60, and 80 g liter⁻¹, maximum ethanol concentrations of 8.8, 10.9, and 9.8 g liter⁻¹ were obtained, respectively (57.2, 57.1, and 48.3% of theoretical). Ethanol was found to inhibit the fermentation of D-xylose (K_p = 0.58 g liter⁻¹) more than the fermentation of glucose (K_p = 6.5 g liter⁻¹). The performance of this yeast compared favorably with that reported for some other D-xylose-fermenting yeasts.     

Multiphasic Kinetics of Transformation of 1,2,4-Trichlorobenzene at Nano- and Micromolar Concentrations by Burkholderia sp. Strain PS14

The transformation of 1,2,4-trichlorobenzene (1,2,4-TCB) at initial concentrations in nano- and micromolar ranges was studied in batch experiments with Burkholderia sp. strain PS14. 1,2,4-TCB was metabolized from nano- and micromolar concentrations to below its detection limit of 0.5 nM. At low initial 1,2,4-TCB concentrations, a first-order relationship between specific transformation rate and substrate concentration was observed with a specific affinity (a⁰_A) of 0.32 liter · mg (dry weight)⁻¹ · h⁻¹ followed by a second one at higher concentrations with an a^o_A of 0.77 liter · mg (dry weight)⁻¹ · h⁻¹. This transition from the first-order kinetics at low initial 1,2,4-TCB concentrations to the second first-order kinetics at higher 1,2,4-TCB concentrations was shifted towards higher initial 1,2,4-TCB concentrations with increasing cell mass. At high initial concentrations of 1,2,4-TCB, a maximal transformation rate of approximately 37 nmol · min⁻¹ · mg (dry weight)⁻¹ was measured, irrespective of the cell concentration.     

Microbial and Physiological Characterization of Weakly Amylolytic but Fast-Growing Lactic Acid Bacteria: a Functional Role in Supporting Microbial Diversity in Pozol, a Mexican Fermented Maize Beverage

Pozol is an acid beverage obtained from the natural fermentation of nixtamal (heat- and alkali-treated maize) dough. The concentration of mono- and disaccharides from maize is reduced during nixtamalization, so that starch is the main carbohydrate available for lactic acid fermentation. In order to provide some basis to understand the role of amylolytic lactic acid bacteria (ALAB) in this fermented food, their diversity and physiological characteristics were determined. Forty amylolytic strains were characterized by phenotypic and molecular taxonomic methods. Four different biotypes were distinguished via ribotyping; Streptococcus bovis strains were found to be predominant. Streptococcus macedonicus, Lactococcus lactis, and Enterococcus sulfureus strains were also identified. S. bovis strain 25124 showed extremely low amylase yield relative to biomass (139 U g [cell dry weight]⁻¹) and specific rate of amylase production (130.7 U g [cell dry weight]⁻¹ h⁻¹). In contrast, it showed a high specific growth rate (0.94 h⁻¹) and an efficient energy conversion yield to bacterial cell biomass (0.31 g of biomass g of substrate⁻¹). These would confer on the strain a competitive advantage and are the possible reasons for its dominance. Transient accumulation of maltooligosaccharides during fermentation could presumably serve as energy sources for nonamylolytic species in pozol fermentation. This would explain the observed diversity and the dominance of nonamylolytic lactic acid bacteria at the end of fermentation. These results are the first step to understanding the importance of ALAB during pozol fermentation.     

Primary and Bacterial Secondary Production in a Southwestern Reservoir

Rates of primary and bacterial secondary production in Lake Arlington, Texas, were determined. The lake is a warm (annual temperature range, 7 to 32°C), shallow, monomictic reservoir with limited macrophyte development in the littoral zone. Samples were collected from six depths within the photic zone from a site located over the deepest portion of the lake. Primary production and bacterial production were calculated from NaH¹⁴CO₃ and [methyl-³H]thymidine incorporation, respectively. Peak instantaneous production ranged between 14.8 and 220.5 μg of C liter⁻¹ h⁻¹. There were two distinct periods of high rates of production. From May through July, production near the metalimnion exceeded 100 μg of C liter⁻¹ h⁻¹. During holomixis, production throughout the water column was in excess of 100 μg of C liter⁻¹ h⁻¹ and above 150 μg of C liter⁻¹ h⁻¹ near the surface. Annual areal primary production was 588 g of C m⁻². Bacterial production was markedly seasonal. Growth rates during late fall through spring were typically around 0.002 h⁻¹, and production rates were typically 5 μg of C liter⁻¹ h⁻¹. Growth rates were higher during warmer parts of the year and reached 0.03 h⁻¹ by August. The maximum instantaneous rate of bacterial production was approximately 45 μg of C liter⁻¹ h⁻¹. Annual areal bacterial production was 125 g of C m⁻². Temporal and spatial distributions of bacterial numbers and activities coincided with temporal and spatial distributions of primary production. Areal primary and bacterial secondary production were highly correlated (r = 0.77, n = 15, P < 0.002).     

Modeling of the pyruvate production with<Emphasis Type="Italic"> Escherichia coli</Emphasis> in a fed-batch bioreactor

A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q_VG=10 mL h^–1. Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q_VG=20 and 30 mL h^–1, respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking–Piret/Levenspiel term).List of symbols c_A acetate concentration (g L^–1) - c_A,0 acetate concentration in the feed (g L^–1) - c_G glucose concentration (g L^–1) - c_G,0 glucose concentration in the feed (g L^–1) - c_P pyruvate concentration (g L^–1) - c_P,max critical pyruvate concentration above which reaction cannot proceed (g L^–1) - c_X biomass concentration (g L^–1) - K_I inhibition constant for pyruvate production (g L^–1) - K_I^A inhibition constant for biomass growth on acetate (g L^–1) - K_P saturation constant for pyruvate production (g L^–1) - K_P inhibition constant of Jerusalimsky (g L^–1) - K_S^A Monod growth constant for acetate (g L^–1) - K_S^G Monod growth constant for glucose (g L^–1) - m_A maintenance coefficient for growth on acetate (g g^–1 h^–1) - m_G maintenance coefficient for growth on glucose (g g^–1 h^–1) - n constant of extended Monod kinetics (Levenspiel) (–) - q_V volumetric flow rate (L h^–1) - q_VA volumetric flow rate of acetate (L h^–1) - q_VG volumetric flow rate of glucose (L h^–1) - r_A specific rate of acetate consumption (g g^–1 h^–1) - r_G specific rate of glucose consumption (g g^–1 h^–1) - r_P specific rate of pyruvate production (g g^–1 h^–1) - r_P,max maximum specific rate of pyruvate production (g g^–1 h^–1) - t time (h) - V reaction (broth) volume (L) - Y_P/G yield coefficient pyruvate from glucose (g g^–1) - Y_X/A yield coefficient biomass from acetate (g g^–1) - Y_X/A,max maximum yield coefficient biomass from acetate (g g^–1) - Y_X/G yield coefficient biomass from glucose (g g^–1) - Y_X/G,max maximum yield coefficient biomass from glucose (g g^–1) - growth associated product formation coefficient (g g^–1) - non-growth associated product formation coefficient (g g^–1 h^–1) - specific growth rate (h^–1) - _max maximum specific growth rate (h^–1)     

Energetics of Syntrophic Propionate Oxidation in Defined Batch and Chemostat Cocultures

Propionate consumption was studied in syntrophic batch and chemostat cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei. The Gibbs free energy available for the H₂-consuming methanogens was <−20 kJ mol of CH₄⁻¹ and thus allowed the synthesis of 1/3 mol of ATP per reaction. The Gibbs free energy available for the propionate oxidizer, on the other hand, was usually >−10 kJ mol of propionate⁻¹. Nevertheless, the syntrophic coculture grew in the chemostat at steady-state rates of 0.04 to 0.07 day⁻¹ and produced maximum biomass yields of 2.6 g mol of propionate⁻¹ and 7.6 g mol of CH₄⁻¹ for S. fumaroxidans and M. hungatei, respectively. The energy efficiency for syntrophic growth of S. fumaroxidans, i.e., the biomass produced per unit of available Gibbs free energy was comparable to a theoretical growth yield of 5 to 12 g mol of ATP⁻¹. However, a lower growth efficiency was observed when sulfate served as an additional electron acceptor, suggesting inefficient energy conservation in the presence of sulfate. The maintenance Gibbs free energy determined from the maintenance coefficient of syntrophically grown S. fumaroxidans was surprisingly low (0.14 kJ h⁻¹ mol of biomass C⁻¹) compared to the theoretical value. On the other hand, the Gibbs free-energy dissipation per mole of biomass C produced was much higher than expected. We conclude that the small Gibbs free energy available in many methanogenic environments is sufficient for syntrophic propionate oxidizers to survive on a Gibbs free energy that is much lower than that theoretically predicted.     

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556

From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45°C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight⁻¹ at D = 0.1 h⁻¹ to 2 U mg of cell dry weight⁻¹ at D = 0.8 h⁻¹. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.