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.The quality of commercial baker’s yeast (Saccharomyces cerevisiae) is determined by many parameters, including storage stability, osmotolerance, freeze-thaw resistance, rehydration resistance of dried yeast, and color. In view of the primary role of baker’s yeast in dough, fermentative capacity (i.e., the specific rate of carbon dioxide production by yeast upon its introduction into dough) is a particularly important parameter (2).In S. cerevisiae, high sugar concentrations and high specific growth rates trigger alcoholic fermentation, even under fully aerobic conditions (6, 18). Alcoholic fermentation during the industrial production of baker’s yeast is highly undesirable, as it reduces the biomass yield on the carbohydrate feedstock. Industrial baker’s yeast production is therefore performed in aerobic, sugar-limited fed-batch cultures. The conditions in such cultures differ drastically from those in the dough environment, which is anaerobic and with sugars at least initially present in excess (23).Optimization of biomass productivity requires that the specific growth rate and biomass yield in the fed-batch process be as high as possible. In the early stage of the process, the maximum feasible growth rate is dictated by the threshold specific growth rate at which respirofermentative metabolism sets in. In later stages, the specific growth rate is decreased to avoid problems with the limited oxygen transfer and/or cooling capacity of industrial bioreactors (10, 27). The actual growth rate profile during fed-batch cultivation is controlled primarily by the feed rate profile of the carbohydrate feedstock (4, 22). Generally, an initial exponential feed phase is followed by phases with constant and declining feed rates, respectively (8).From a theoretical point of view, the objective of suppressing alcoholic fermentation during the production phase may interfere with the aim of obtaining a high fermentative capacity in the final product. Process optimization has so far been based on strain selection and on empirical optimization of environmental conditions during fed-batch cultivation (e.g., pH, temperature, aeration rate, and feed profiles of sugar, nitrogen, and phosphorus [5, 10, 23]). For rational optimization of the specific growth rate profile, knowledge of the relation between specific growth rate and fermentative capacity is of primary importance. Nevertheless, quantitative data on this subject cannot be found in the literature.The chemostat cultivation system allows manipulation of the specific growth rate (which is equal to the dilution rate) while keeping other important growth conditions constant. Similar to industrial fed-batch cultivation, sugar-limited chemostat cultivation allows fully respiratory growth of S. cerevisiae on sugars (21, 37, 39). This is not possible in batch cultures, which by definition require high sugar concentrations, which lead to alcoholic fermentation, even during aerobic growth (6, 18, 37). Thus, as an experimental system, batch cultures bear little resemblance to the aerobic baker’s yeast production process. Indeed, we have recently shown that differences in fermentative capacity between a laboratory strain of S. cerevisiae and an industrial strain became apparent only in glucose-limited chemostat cultures but not in batch cultures (30).The aim of the present study was to assess the effect of specific growth rate on fermentative capacity in an industrial baker’s yeast strain grown in aerobic, sugar-limited chemostat cultures. Furthermore, the effect of specific growth rate on in vitro activities of key glycolytic and fermentative enzymes was investigated in an attempt to identify correlations between fermentative capacity and enzyme levels.     

Intermediary Metabolite Concentrations in Xylulose- and Glucose-Fermenting Saccharomyces cerevisiae Cells

Glucose and xylulose fermentation and product formation by Saccharomyces cerevisiae were compared in batch culture under anaerobic conditions. In both cases the main product was ethanol, with glycerol, xylitol, and arabitol produced as by-products. During glucose and xylulose fermentation, 0.74 and 0.37 g of cell mass liter⁻¹, respectively, were formed. In glucose-fermenting cells, the carbon balance could be closed, whereas in xylulose-fermenting cells, about 25% of the consumed sugar carbon could not be accounted for. The rate of sugar consumption was 3.94 mmol g of initial biomass⁻¹ h⁻¹ for glucose and 0.39 mmol g of initial biomass⁻¹ h⁻¹ for xylulose. Concentrations of the intermediary metabolites fructose-1,6-diphosphate (FDP), pyruvate (PYR), sedoheptulose 7-phosphate (S7P), erytrose 4-phosphate, citrate (CIT), fumarate, and malate were compared for both types of cells. Levels of FDP, PYR, and CIT were lower, and levels of S7P were higher in xylulose-fermenting cells. After normalization to the carbon consumption rate, the levels of FDP were approximately the same, whereas there was a significant accumulation of S7P, PYR, CIT, and malate, especially of S7P, in xylulose-fermenting cells compared with in glucose-fermenting cells. In the presence of 15 μM iodoacetate, an inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), FDP levels increased and S7P levels decreased in xylulose-assimilating cells compared with in the absence of the inhibitor, whereas fermentation was slightly slowed down. The specific activity of transaldolase (EC 2.2.1.2), the pentose phosphate pathway enzyme reacting with S7P and glyceraldehyde-3-phosphate, was essentially the same for both glucose- and xylulose-fermenting cells. It was, however, several orders of magnitude lower than that reported for a Torula yeast and Candida utilis. The presence of iodoacetate did not influence the activity of transaldolase in xylulose-fermenting cells. The results are discussed in terms of a competition between the pentose phosphate pathway and glycolysis for the common metabolite, glyceraldehyde-3-phosphate, which would explain the low rates of xylulose assimilation and ethanol production from xylulose by S. cerevisiae.     

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.     

Continuous and Batch Production of Chloroperoxidase by Mycelial Pellets of Caldariomyces fumago in an Airlift Fermentor

Batch and continuous production of the extracellular heme glycoprotein chloroperoxidase (CPO) was studied with an airlift fermentor. We induced Caldariomyces fumago CMI 89362 to form pellets by transferring a small inoculum volume in preculture prior to growth in a 1-liter fermentor. Continuous replacement of the fructose-salts medium (dilution rate, 0.008 h⁻¹) supported continuous CPO formation at an average concentration of 128 ± 10 mg of CPO liter⁻¹ for 8 days. Optimum CPO production rates averaged 1.2 ± 0.1 mg of CPO h⁻¹ at dilution rates below 0.033 h⁻¹. Varying the carbohydrate content of the feed solution or the time of starting the feed did not significantly alter the amount of CPO produced. Batch fermentation in the airlift fermentor resulted in maximum CPO concentrations of 280 ± 80 mg of CPO liter⁻¹, although on two separate occasions CPO concentrations reached 400 to 450 mg liter⁻¹, which was double the amount obtained by free hyphae in shake flask culture.     

Perfusion Method for Assaying Microbial Activities in Sediments: Applicability to Studies of N2 Fixation by C2H2 Reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol · g of dry sediment⁻¹ · h⁻¹ by 10 to 20 h. Depletion of interstitial NH₄⁺ was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C₂H₄ production. Initial values obtained by using the perfusion method were 0.66 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Zostera communities and 0.70 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

Kinetics of Perchlorate- and Chlorate-Respiring Bacteria

Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated “Dechlorosoma” sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 h⁻¹, respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 h⁻¹, respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 h⁻¹, respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h⁻¹, respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 h⁻¹) and exceeded that of strain KJ on perchlorate (0.14 h⁻¹). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 ± 0.07 [n = 7]), chlorate (0.44 ± 0.05 [n = 7]), and perchlorate (0.50 ± 0.08 [n = 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors.     

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.     

Formation of Hydride-Meisenheimer Complexes of Picric Acid (2,4,6-Trinitrophenol) and 2,4-Dinitrophenol during Mineralization of Picric Acid by Nocardioides sp. Strain CB 22-2

There are only a few examples of microbial conversion of picric acid (2,4,6-trinitrophenol). None of the organisms that have been described previously is able to use this compound as a sole source of carbon, nitrogen, and energy at high rates. In this study we isolated and characterized a strain, strain CB 22-2, that was able to use picric acid as a sole source of carbon and energy at concentrations up to 40 mM and at rates of 1.6 mmol · h⁻¹ · g (dry weight) of cells⁻¹ in continuous cultures and 920 μmol · h⁻¹ · g (dry weight) of cells⁻¹ in flasks. In addition, this strain was able to use picric acid as a sole source of nitrogen at comparable rates in a nitrogen-free medium. Biochemical characterization and 16S ribosomal DNA analysis revealed that strain CB 22-2 is a Nocardioides sp. strain. High-pressure liquid chromatography and UV-visible light data, the low residual chemical oxygen demand, and the stoichiometric release of 2.9 ± 0.1 mol of nitrite per mol of picric acid provided strong evidence that complete mineralization of picric acid occurred. During transformation, the metabolites detected in the culture supernatant were the [H⁻]-Meisenheimer complexes of picric acid and 2,4-dinitrophenol (H⁻-DNP), as well as 2,4-dinitrophenol. Experiments performed with crude extracts revealed that H⁻-DNP formation indeed is a physiologically relevant step in picric acid metabolism.     

Chemostat Production of Plantaricin C By Lactobacillus plantarum LL441

Plantaricin C, a bacteriocin synthesized by Lactobacillus plantarum LL441, was optimally produced in chemostats kept at pH 5.0, 30°C, 150 rpm, and a dilution rate of 0.05 h⁻¹ when glucose was used as carbon source and a dilution rate of 0.10 to 0.12 h⁻¹ when sucrose or fructose was used instead. Production was abolished at high dilution rates, i.e., when the cells grew rapidly in all carbon sources.     

Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h⁻¹ g (dry weight) of cells⁻¹ (0.24 to 0.30 g h⁻¹ g [dry weight] of cells⁻¹) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h⁻¹. The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.     

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.     

Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min⁻¹. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h⁻¹ compared with 0.117 h⁻¹) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Effect of Growth Rate and Starvation-Survival on the Viability and Stability of a Psychrophilic Marine Bacterium

Cell populations of the marine bacterium ANT-300, from either batch or continuous culture with dilution rates ranging from D = 0.015 h⁻¹ to D = 0.200 h⁻¹, were monitored for viability, direct counts, and optical density for 98 days under starvation conditions. Three stages of starvation survival were observed for each of the cell populations. Although direct counts remained at 2 × 10⁷ to 3 × 10⁷ cells ml⁻¹ throughout the starvation period, large fluctuations occurred in cell viability during stage 1 (0 to 14 days) of starvation survival. Stage 2 (14 to 70 days) involved an overall decrease in viability for each of the cell populations; the rate of viability loss was dependent upon the growth rate. Cell viability stabilized at approximately 0.3% of the direct count in stage 3 (70 to 98 days). Long-term starvation corresponded to the prolongation of stage 3 starvation survival. Cell volumes for each of the cell populations decreased with the length of the starvation period. However, the cell volume of starved cells was also dependent more on growth rate than on the length of the time starved. We hypothesize that the cell population with the slowest growth rate is most closely representative of cells found in the oligotrophic marine environment.     

Structure-Activity Relationships in Microbial Transformation of Phenols

The second-order rate constants for the microbial transformation of a series of phenols were correlated with the physicochemical properties of the phenols. The compounds studied were phenol, p-methylphenol, p-chlorophenol, p-bromophenol, p-cyanophenol, p-nitrophenol, p-acetylphenol, and p-methoxyphenol. Phenol-grown cells of Pseudomonas putida U transformed these compounds. Microbial transformation rate constants ranged from (1.5 ± 0.99) × 10⁻¹⁴ liter · organism⁻¹ · h⁻¹ for p-cyanophenol to (7.0 ± 1.3) × 10⁻¹² liter · organism⁻¹ · h⁻¹ for phenol. Linear regression analyses of rate constants and electronic, steric, and hydrophobic parameters showed that van der Waal's radii gave the best coefficient of determination (r² = 0.956). Products identified by thin-layer chromatography and liquid chromatography indicated that the phenols were microbially oxidized to the corresponding catechols.     

Kinetic Constant Variability in Bacterial Oxidation of Elemental Sulfur

Wide ranges of growth yields on sulfur (from 2.4 × 10¹⁰ to 8.1 × 10¹¹ cells g⁻¹) and maximum sulfur oxidation rates (from 0.068 to 1.30 mmol liter⁻¹ h⁻¹) of an Acidithiobacillus ferrooxidans strain (CCM 4253) were observed in 73 batch cultures. No significant correlation between the constants was observed. Changes of the Michaelis constant for sulfur (from 0.46 to 15.5 mM) in resting cells were also noted.     

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.     

Ethanol Production from Colpomenia sinuosa by an Alginate Fermentation Strain Meyerozyma guilliermondii

With the consumption of energy and the spread of COVID-19, the demand for ethanol production is increasing in the world. The industrial ethanol fermentation microbes cannot metabolize the alginate component of macro algae, which affects the ethanol yield. In this research, the ethanol production process from macro algae by an alginate fermentation yeast Meyerozyma guilliermondii, especially the pretreatment process of Colpomenia sinuosa, was studied. At the same time, the experimental design of Box-Behnken was carried out to achieve the optimum fermentation performance. The concentration of KH₂PO₄ (A: 2–6 g.L⁻¹), pH (B: 4–7), reaction time (C: 60–120 h) and temperature (D: 24–34 °C) were variable input parameters. During the ethanol production process, the algae powder was firstly mixed with water at 90 °C for 0.5 h. Later the fermentation culture medium was prepared and then it was fermented by the yeast Meyerozyma guilliermondii to produce ethanol. And the optimal fermentation parameters were as follows: fermentation temperature of 28 °C, KH₂PO₄ dosage of 4.7 g.L⁻¹, initial pH of 6, and fermentation time of 99 h. The ethanol yield reached 0.268 g.g⁻¹ (ethanol to algae), close to the predicted value of model. The generation of alginate lyase during the fermentation of algae was also examined. The highest alginate lyase activity reached 46.42 U.mL⁻¹.     

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.     

Volatile Fatty Acid Production by the Hindgut Microbiota of Xylophagous Termites

Acetate dominated the extracellular pool of volatile fatty acids (VFAs) in the hindgut fluid of Reticulitermes flavipes, Zootermopsis angusticollis, and Incisitermes schwarzi, where it occurred at concentrations of 57.9 to 80.6 mM and accounted for 94 to 98 mol% of all VFAs. Small amounts of C₃ to C₅ VFAs were also observed. Acetate was also the major VFA in hindgut homogenates of Schedorhinotermes lamanianus, Prorhinotermes simplex, Coptotermes formosanus, and Nasutitermes corniger. Estimates of in situ acetogenesis by the hindgut microbiota of R. flavipes (20.2 to 43.3 nmol · termite⁻¹ · h⁻¹) revealed that this activity could support 77 to 100% of the respiratory requirements of the termite (51.6 to 63.6 nmol of O₂ · termite⁻¹ · h⁻¹). This conclusion was buttressed by the demonstration of acetate in R. flavipes hemolymph (at 9.0 to 11.6 mM), but not in feces, and by the ability of termite tissues to readily oxidize acetate to CO₂. About 85% of the acetate produced by the hindgut microbiota was derived from cellulose C; the remainder was derived from hemicellulose C. Selective removal of major groups of microbes from the hindgut of R. flavipes indicated that protozoa were primarily responsible for acetogenesis but that bacteria also functioned in this capacity. H₂ and CH₄ were evolved by R. flavipes (usually about 0.4 nmol · termite⁻¹ · h⁻¹), but these compounds represented a minor fate of electrons derived from wood dissimilation within R. flavipes. A working model is proposed for symbiotic wood polysaccharide degradation in R. flavipes, and the possible roles of individual gut microbes, including CO₂-reducing acetogenic bacteria, are discussed.     

DIFFERENCES IN GROUND CONTACT TIME EXPLAIN THE LESS EFFICIENT RUNNING ECONOMY IN NORTH AFRICAN RUNNERS

The purpose of this study was to investigate the relationship between biomechanical variables and running economy in North African and European runners. Eight North African and 13 European male runners of the same athletic level ran 4-minute stages on a treadmill at varying set velocities. During the test, biomechanical variables such as ground contact time, swing time, stride length, stride frequency, stride angle and the different sub-phases of ground contact were recorded using an optical measurement system. Additionally, oxygen uptake was measured to calculate running economy. The European runners were more economical than the North African runners at 19.5 km · h⁻¹, presented lower ground contact time at 18 km · h⁻¹ and 19.5 km · h⁻¹ and experienced later propulsion sub-phase at 10.5 km · h⁻¹,12 km · h⁻¹, 15 km · h⁻¹, 16.5 km · h⁻¹ and 19.5 km · h⁻¹ than the European runners (P < 0.05). Running economy at 19.5 km · h⁻¹ was negatively correlated with swing time (r = -0.53) and stride angle (r = -0.52), whereas it was positively correlated with ground contact time (r = 0.53). Within the constraints of extrapolating these findings, the less efficient running economy in North African runners may imply that their outstanding performance at international athletic events appears not to be linked to running efficiency. Further, the differences in metabolic demand seem to be associated with differing biomechanical characteristics during ground contact, including longer contact times.