Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

During glucose‐limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β‐lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady‐state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose‐limited chemostat cultures at the dilution rates (D) of both 0.03 h^?1 and 0.05 h^?1 are determined to evaluate the quantitative impact on the physiology of a high‐yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h^?1, while this is not the case at D = 0.05 h^?1. In addition, a higher cytosolic redox status (NADH/NAD⁺), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h^?1, which are virtually absent at D = 0.03 h^?1. In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose‐limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial‐scale scenario.     

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

Effect of dilution rate and carbon availability on Bifidobacterium breve fermentation

Bifidobacterium breve NCFB 2257 was grown in glucose-limited and nitrogen (N)-limited chemostats at dilution rates (D) from 0.04 to 0.60 h^–1, to study the effect of nutrient availability on carbohydrate metabolism. The results showed that D had little effect on fermentation product formation, irrespective of the form of nutrient limitation. However, marked differeces were observed in the distribution of fermentation products, that were attributable to glucose availability. In glucose-limited cultures, formate and acetate were the principal end-products of metabolism. Lactate was never detected under these growth conditions. In contrast, lactate and acetate were mainly formed when glucose was in excess, and formate was not produced. These results are explained by the metabolic fate of pyruvate, which can be dissimilated by either phosphoroclastic cleavage to acetyl phosphate and formate, or alternatively, it may be reduced to lactate. Enzymic studies were made to establish the mechanisms that regulated pyruvate metabolism. The data demonstrated that control was not exercised through regulation of the synthesis and activity of lactate dehydrogenase (LDH), phosphofructokinase or alcohol dehydrogenase. It is possible however, that there was competition for pyruvate by LDH and the phosphoroclastic enzyme, which would determine the levels of lactate and formate produced respectively. These results demonstrate the metabolic flexibility of B. breve, which preferentially uses lactate as an electron sink during N-limited growth, whereas under energy-limitation, carbon flow is directed towards acetyl phosphate to maximise ATP synthesis. Correspondence to: B. A. Degnan     

Regulation of Escherichia coli formate hydrogenlyase activity by formate at alkaline pH

Escherichia coli possesses two hydrogenases, Hyd-3 and Hyd-4. These, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenlyases, FHL-1 and FHL-2, both catalyzing the decomposition of formate to H₂ and CO₂ during fermentative growth. FHL-1 is the major pathway at acidic pH whereas FHL-2 is proposed for slightly alkaline pH. In this study, regulation of activity of these pathways by formate has been investigated. In cells grown under fermentative conditions on glucose in the presence of 30 mM formate at pH 7.5, intracellular pH was decreased to 7.1, the activity of Fdh-H raised 3.5-fold, and the production of H₂ became mostly Hyd-3 dependent. These results suggest that at alkaline pH formate increases an activity of Fdh-H and of Hyd-3 both but not of Hyd-4. Received: 27 December 2001 / Accepted: 25 January 2002     

Growth yield and energy generation in anaerobically-grown Campylobacter spec.

An anaerobic continuous culture study was made with Campylobacter spec. to determine growth yields under various growth conditions. The growth media contained 0.1% (w/v) yeast extract as carbon source. When grown in an aspartate-limited culture Y _asp ^max was 4.6. Inclusion of formate in the culture medium hardly affected the true growth yield. The number of ATP equivalents generated in the fumaratereductase system was 0.66 and the Y _ATP ^max was 7.0. In the nitrate reduction with formate 1.7 ATP equivalents were generated, and a YNO _3- ^max of 12.2 was observed. The true growth yield obtained with a mixture of lactate and aspartate was lower than that found with aspartate alone.     

Inhibition of Cathepsin B1 by E-64, a Thiol Proteinase Inhibitor,and Its Derivatives

Four formaldehyde-resistant yeasts were isolated from soil. Three were tentatively identified as Debaryomyces vanriji and one as Trichosporon penicillatum. These yeasts almost completely consumed formaldehyde at 0.15 to 0.55% in growth medium containing glucose as carbon source, but the carbon of formaldehyde was not incorporated into the cell constituents. In formaldehyde-containing medium, yeast growth occurred after formaldehyde consumption. The yeasts showed relatively high activities of formaldehyde dehydrogenase, S-formylglutathione hydrolase and formate dehydrogenase. The resistance to formaldehyde is attributed to detoxification by oxidation.     

Effects of Formate on Fermentative Hydrogen Production by Enterobacter aerogenes

This paper describes the effects of formate on fermentative hydrogen production by Enterobacter aerogenes by way of batch culture. When 20 mM formate was added to pH 6.3 and pH 5.8 E. aerogenes glucose cultures (formate culture) at the beginning of cultivation, hydrogen evolution through both glucose consumption and decomposition of the extrinsic formate occurred together, while hydrogen evolution occurred only through glucose consumption in the control cultures. The hydrogen evolution rates in the formate cultures were faster than in the control cultures, although cell growth and glucose consumption rates in the formate cultures were slower than the control cultures’. The decomposition rate of the extrinsic formate in the pH 5.8 formate culture was faster than in the pH 6.3 fomiate culture. The hydrogen yield from glucose in the pH 6.3 formate culture increased due to the increasing amount of the nicotinamide adenine dinucleotide for hydrogen production.     

Characterization of optimal growth conditions and carotenoid production of strain Rhodotorula mucilaginosa HP isolated from larvae of Pieris rapae

Yeasts have been studied because of their production of a pigment known as carotenoid with potential application in food and feed supplements. A carotenoid‐producing yeast was isolated from the larvae of Pieris rapae, named HP. The strain HP was identified as Rhodotorula mucilaginosa classified by its carbohydrate fermentation pattern and physiological tests. Rhodotorula mucilaginosa HP produces several exogenous enzymes: alkaline phosphatase, esterase, leucine arylamidase, valine arylamidase, acid phosphatase and β‐glucosidase. Using response surface methodology, selected medium components (yeast extract, malt extract, peptone, glucose) were tested to find the optimum conditions for carotenoid production and the growth of R. mucilaginosa HP. Central composite design was used to control the concentrations of medium components. Peptone and glucose had the largest effects on carotenoid production and cell growth of R. mucilaginosa HP, respectively. The estimated optimal growth conditions of R. mucilaginosa HP were: yeast extract 3.23%, malt extract 2.84%, peptone 6.99% and glucose 12.86%. The estimated optimal conditions for carotenoid production were: yeast extract 2.17%, malt extract 2.11%, peptone 5.79% and glucose 12.46%. These results will assist in the formulation of an appropriate culture medium for optimal carotenoid production of R. mucilaginosa HP for commercial use.     

Effect of tungsten and molybdenum on growth of a syntrophic coculture of <Emphasis Type="Italic">Syntrophobacter fumaroxidans</Emphasis> and <Emphasis Type="Italic">Methanospirillum hungatei</Emphasis>

The effect of tungsten (W) and molybdenum (Mo) on the growth of Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied in syntrophic cultures and the pure cultures of both the organisms. Cells that were grown syntropically were separated by Percoll density centrifugation. Measurement of hydrogenase and formate dehydrogenase levels in cell extracts of syntrophically grown cells correlated with the methane formation rates in the co-cultures. The effect of W and Mo on the activity of formate dehydrogenase was considerable in both the organisms, whereas hydrogenase activity remained relatively constant. Depletion of tungsten and/or molybdenum, however, did not affect the growth of the pure culture of S. fumaroxidans on propionate plus fumarate significantly, although the specific activities of hydrogenase and especially formate dehydrogenase were influenced by the absence of Mo and W. This indicates that the organism has a low W or Mo requirement under these conditions. Growth of M. hungatei on either formate or H₂/CO₂ required tungsten, and molybdenum could replace tungsten to some extent. Our results suggest a more prominent role for H₂ as electron carrier in the syntrophic conversion of propionate, when the essential trace metals W and Mo for the functioning of formate dehydrogenase are depleted.     

Increased product formation induced by a directed secondary substrate limitation in a batch Hansenula polymorpha culture

By the use of directed limitations of secondary substrates, the metabolic flux should be deflected from biomass production to product formation. In order to study the impact of directed limitations caused by various secondary substrates on the growth and product formation of the methylotrophic yeast Hansenula polymorpha, the cultivation systems respiration activity monitoring system (RAMOS) and BioLector were used in parallel. While the RAMOS device allows the online monitoring of the oxygen transfer rate in shake flasks, the BioLector enables in microtiter plates the monitoring of scattered light and the fluorescence intensity of the green fluorescent protein (GFP). Secondary substrate limitations of phosphate, potassium, and magnesium were analyzed in batch fermentations. The sole carbon source was either 10 g/L glucose or 10 g/L glycerol. The expression of the GFP gene is controlled by the FMD promoter (formate dehydrogenase). In batch cultures with glucose as carbon source, a directed limitation of phosphate increased the GFP production 1.87-fold, compared to phosphate unlimited conditions. Under potassium-limited conditions with glycerol as sole carbon source, the GFP production was 1.41-fold higher compared to unlimited conditions. A limitation of the substrate magnesium resulted in a 1.22-fold increase GFP formation in the case of glycerol as carbon source.     

Anoxybacillus kamchatkensis sp. nov., a novel thermophilic facultative aerobic bacterium with a broad pH optimum from the Geyser valley, Kamchatka

A facultative aerobic, moderately thermophilic, spore forming bacterium, strain JW/VK-KG4 was isolated from an enrichment culture obtained from the Geyser valley, a geothermally heated environment located in the Kamchatka peninsula (Far East region of Russia). The cells were rod shaped, motile, peritrichous flagellated stained Gram positive and had a Gram positive type cell wall. Aerobically, the strain utilized a range of carbohydrates including glucose, fructose, trehalose, proteinuous substrates, and pectin as well. Anaerobically, only carbohydrates are utilized. When growing on carbohydrates, the strain required yeast extract and vitamin B₁₂. Anaerobically, glucose was fermented to lactate as main product and acetate, formate, ethanol as minor products. Aerobically, even in well-aerated cultures (agitated at 500 rpm), glucose oxidation was incomplete and lactate and acetate were found in culture supernatants as by-products. Optimal growth of the isolate was observed at pH^{25 C} 6.8–8.5 and 60°C. The doubling times on glucose at optimal growth conditions were 34 min (aerobically) and 40 min (anaerobically). The G+C content was 42.3 mol% as determined by T_m assay. Sequence analysis of the 16S rRNA gene indicated an affiliation of strain JW/VK-KG4 with Anoxybacillus species. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA homology with validly published species of Anoxybacillus, it is proposed that strain JW/VK-KG4 represents a new species in the genus Anoxybacillus as A. kamchatkensis sp. nov. The type strain for the novel species is JW/VK-KG4^T (=DSM 14988, =ATCC BAA-549). The GenBank accession number for the 16S rDNA sequence is AF510985.     

Coordinated Development of Yeast Colonies: Quantitative Modeling of Diffusion‐Limited Growth – Part 2

A mathematical model was developed which described the growth of yeast colonies based on the assumptions that (i) these populations were built up of single cells whose proliferation was (ii) exclusively controlled by nutrient availability in the environment. The model was of a hybrid cellular automaton type and described discrete cells residing on a one‐dimensional lattice as well as on continuously distributed nutrients. Experimental results and numerical calculations were compared to elucidate under which cultivation conditions the diffusion‐limited growth (DLG) was the major construction principle in yeast colonies. Simulations were scaled to the growth of Yarrowia lipolytica and Candida boidinii colonies under carbon and nitrogen limitation. They showed that nutrient‐controlled growth of the individual cells resulted in DLG of the population. Quantitative predictions for the spatio‐temporal development of the cell‐density profile inside a growing yeast mycelium were compared to the growth characteristics of the model yeast mycelia. Only for the carbon‐limited growth of C. boidinii colonies on glucose as the limiting nutrient resource did the DLG model reproduce the cell‐density profile estimated at the end of the cultivation. Under all other cultivation conditions, strong discrepancies between calculations and experimental results were evident precluding DLG as the ruling regulatory mechanism. Thus, whether or not the development of a yeast population could be described by a DLG scenario, was strongly dependent on the particular cultivation conditions and the applied yeast species. In those cases for which the DLG hypothesis failed to explain the observed growth patterns, the underlying assumptions, i.e., the complete absence of nutrient translocation between the individual cells inside the yeast mycelia as well as the exclusively nutrient‐controlled proliferation of the cells, have to be reevaluated. The presented study demonstrated how the mathematical analysis of growth processes in yeast populations could assist the experimental identification of potential regulatory mechanisms.     

The role of hexokinase in plant sugar signal transduction and growth and development

Previous studies have revealed a central role of Arabidopsis thaliana hexokinases (AtHXK1 and AtHXK2) in the glucose repression of photosynthetic genes and early seedling development. However, it remains unclear whether HXK can modulate the expression of diverse sugar-regulated genes. On the basis of the results of analyses of gene expression in HXK transgenic plants, we suggest that three distinct glucose signal transduction pathways exist in plants. The first is an AtHXK1-dependent pathway in which gene expression is correlated with the AtHXK1-mediated signaling function. The second is a glycolysis-dependent pathway that is influenced by the catalytic activity of both AtHXK1 and the heterologous yeast Hxk2. The last is an AtHXK1-independent pathway in which gene expression is independent of AtHXK1. Further investigation of HXK transgenic Arabidopsis discloses a role of HXK in glucose-dependent growth and senescence. In the absence of exogenous glucose, plant growth is limited to the seedling stage with restricted true leaf development even after a 3-week culture on MS medium. In the presence of glucose, however, over-expressing Arabidopsis or yeast HXK in plants results in the repression of growth and true leaf development, and early senescence, while under-expressing AtHXK1 delays the senescence process. These studies reveal multiple glucose signal transduction pathways that control diverse genes and processes that are intimately linked to developmental stages and environmental conditions.     

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.     

The effect of mixtures of glucose and formate on the yield and respiration of a chemostat culture of Beneckea natriegens

Beneckea natriegens oxidizes sodium formate constitutively when grown on glucose or glycerol in chemostat culture, but cannot utilize formate as the sole source of carbon and energy for growth. However, when grown on a mixture of glucose and formate (D=0.37 h^-1, pH 7.6) the yield is higher than on glucose alone.The yield, expressed in terms of g bacterial dry weight g^-1 glucose plus formate carbon utilized, gave a linear relationship when plotted against the total heat of combustion of glucose plus formate utilized. Extrapolation of the plot cut the abscissa at a value equivalent to the heat of combustion of formate, which suggests that formate is not utilised as a source of carbon but only energy.In cultures with nitrate as the sole source of nitrogen the yield from glucose was lower than that observed with ammonia but the addition of formate to the culture utilizing nitrate resulted in an increase in the yield from glucose to a value similar to that observed with ammonia.At a culture pH value of 7.65 unused formate (<0.15–227 mM) in the culture supernatant had no effect on respiration spiration or yield, but at a culture pH of 6.7 excess formate caused a marked increase in respiration rate and a large decrease in the yield from glucose; further decrease in the pH value caused washout of the culture. This may be explained by undissociated formic acid causing uncoupling of oxidative phosphorylation.     

Growth yield increase linked to caffeate reduction in Acetobacterium woodii

Growth yields were determined with Acetobacterium woodii strain NZva 16 on hydrogen and CO₂, formate, methanol, vanillate, ferulate and fructose in mineral medium in the absence and presence of 0.05% yeast extract. Yeast extract was not essential for growth but enhanced growth yields by 25–100% depending on the substrate fermented. Comparison of yields on formate or methanol allowed calculation of an energy yield in the range of 1.5–2 mol ATP per mol acetate formed during homoacetate fermentation of A. woodii. In the presence of 6 mM caffeate, growth yields were determined with the substrates formate or methanol. Caffeate was reduced to hydrocaffeate and increased growth yields were obtained. An ATP yield of about 1 mol per mol of caffeate reduced was calculated. Cytochromes were not detectable in cell free extracts or membrane preparations.     

Determination of Growth and Glycerol Production Kinetics of a Wine Yeast Strain Saccharomyces cerevisiae Kalecik 1 in Different Substrate Media

Summary Glycerol has been known as an important by-product of wine fermentations improving the sensory quality of wine. This study was carried out with an endogenic wine yeast strain Saccharomyces cerevisiae Kalecik 1. The kinetics of growth and glycerol biosynthesis were analysed at various initial concentrations of glucose, fructose, and sucrose in a batch system. Depending on the determined values of Monod constants, glucose (K_s = 28.09 g/l) was found as the most suitable substrate for the yeast growth. Initial glucose, fructose and sucrose concentrations necessary for maximum specific yeast growth rate were determined as 175 g, 100 l, and 200 g/l, respectively. The yeast produced glycerol at very high concentrations in fructose medium. Fructose was determined as the most suitable substrate for glycerol production while the strain showed low tendency to use it for growth. S. cerevisiae Kalecik 1 could not produce glycerol below 200 g/l initial sucrose concentration. When natural white grape juice was used as fermentation medium, maximum glycerol concentration and dry weight of the yeast were determined as 9.3 g/l and 11.8 g/l, respectively.     

Overexpression of formate dehydrogenase inArabidopsis thaliana resulted in plants tolerant to high concentrations of formate

The potential role played by formate dehydrogenase (FDH) in formate metabolism has been examined by the overexpression of FDH in Arabidopsis thaliana. Three independent transgenic lines were selected and shown to produce elevated amounts of FDH protein with a corresponding elevated FDH activity (2.5-5 fold) over wild-type (WT) plants. Under normal growth conditions, no altered phenotype was observed in these transgenic plants; in growth media supplied with formate, however, significant differences in shoot and root growth, compared to that of WT plants, were observed. WT plants were severely injured if grown in the presence of 16 mmol/L formate, while the transgenic plants were able to grow well. Formate delayed germination of both WT and transgenic seeds at concentrations above 4 mmol/L, but both types of seeds were eventually able to complete more than 95 % germination even at 32 mmol/L formate. Formate markedly inhibited primary root elongation, and its inhibitory action on WT was much stronger than on transgenic plants. Different formate salts affected root elongation similarly, indicating that the formate ion was the major factor inhibiting root growth. Sodium acetate (NaAc), an analogue of formate, also inhibited root elongation, but its action on WT and transgenic plants was the same, indicating that tolerance of transgenic plants to formate toxicity was specific. Transgenic plants showed no significant tolerance to the toxicity of two other one-carbon metabolites, methanol and formaldehyde. A role for FDH in detoxifying formate is proposed.     

Glucose Fermentation by <Emphasis Type="Italic">Propionibacterium microaerophilum</Emphasis>: 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, CO₂, 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 CO₂ 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. Received: 8 April 2002 / Accepted: 8 May 2002     

硝酸盐对牛粪梭菌甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响

【背景】异于同型产乙酸菌通常利用Wood-Ljungdahl途径将2分子CO₂还原为1分子乙酰辅酶A,Clostridium bovifaecis缺失Wood-Ljungdahl途径甲基支路第1步将CO₂还原为甲酸的甲酸脱氢酶,需甲酸存在时将1分子甲酸和1分子CO₂还原为乙酰辅酶A发生葡萄糖的同型产乙酸型发酵。已有报道显示,硝酸盐也可作为同型产乙酸菌的电子受体,而且对不同同型产乙酸菌的代谢影响有所不同,然而硝酸盐对这种独特的甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响尚不清楚。【目的】探究硝酸盐对C.bovifaecis甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响。【方法】硝酸盐浓度分别为10 mmol/L和30 mmol/L时,以未添加硝酸盐为对照实验,研究C.bovifaecis在葡萄糖+甲酸+CO₂为基质条件下的细菌生长、底物消耗和产物生成情况。【结果】10 mmol/L和30 mmol/L硝酸盐存在时,主要产物乙醇浓度分别为5.80 mmol/L和1.66 mmo...