The influence of acetic acid on penicillin production

Summary Fed-batch fermentations with Penicillum chrysogenum, strain S 3723, were fed with glucose as carbon source or with a mixture of glucose and acetic acid. When 20% of the carbon source was acetic acid, yields of penicillin-V were 25% higher than in fermentations where glucose was the only carbon source in the feed. The increased yield was due to higher specific productivity and/or cell mass. The effect was seen in fermentations where the carbon source was fed at a constant rate and the pH kept automatically at 6.5 by addition of inorganic acid or base, as well as in fermentations where pH controlled the addition of feed.     

Nutrient effects on anthracycline production by Streptomyces peucetius in a defined medium

A defined medium was developed for Streptomyces peucetius that optimally contained 0.5 mM magnesium, 1 mM phosphate, 75-125 mM glucose, 10 mM nitrate, and microelements. Poorer results were obtained with nitrite, aspartate, or ammonia as sole nitrogen sources. Other carbon sources which supported best growth and highest anthracycline titers were fructose, maltose, and soluble starch. In each case, substantial residual carbon remained at the end of 6 days, suggesting a lack of catabolite repression by the carbohydrate carbon sources on anthracycline biosynthesis. Studies involving limiting and nonlimiting concentrations of glucose supplemented with arabinose, a poorly utilizable carbon source, indicated that high carbon concentrations were not necessary for osmotic stabilization. Inorganic phosphate was found to have an inhibitory effect on anthracycline production. Furthermore, when cultures at early stages of anthracycline production were spiked with inorganic phosphate, a delay in further anthracycline production resulted until the added phosphate was depleted. A 10% inoculum of stationary phase cells yielded the best growth and most consistent anthracycline production. Spectrophotometric analyses at 495 nm of chloroform--methanol-extracted material were also found to be useful for the determination of total anthracyclines in culture extracts.     

Effect of cell concentration on the rheology of glucoamylase fermentation broth

During the process optimisation of glucoamylase production by Aspergillus awamori, cell morphology was controlled at such a state that spore aggregation was completely prevented. Samples from five fermentations on complex media using either glucose or starch as carbon source were characterised with a Bohlin CS rheometer. The experimental data were conveniently described in terms of the power law model.     

Conversion of starch into ethanol by Clostridium thermohydrosulfuricum

Summary Production of ethanol from starch by Clostridium thermohydrosulfuricum was compared with that from glucose, fructose or maltose in batch fermentations. Optimal substrate concentration and pH for ethanol production were determined. The rate of ethanol production on starch was about the same as that on glucose or fructose and overall yields were also similar (about 1.6 mol ethanol per mol glucose or glucose equivalent). Maltose was not an effective substrate for growth and ethanol production.When a mixture of starch and glucose in equal amounts was used, breakdown of starch and utilization of glucose were simultaneous. When starch and fructose were supplied together, the fructose was utilized but no hydrolysis of starch was observed. With a mixture of glucose and fructose, uptake of fructose preceeded that of glucose.     

Repeated-batch fermentations of xylose and glucose-xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.     

Some observations on protease production in continuous suspension cultures of Bacillus firmus

Invariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures of Bacillus firmus NRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures of B. firmus over prolonged period is demonstrated in this study. (c) 1993 John Wiley & Sons, Inc.     

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.     

Sequential processes of phosphate limitation and of phosphate release in streptomycin fermentations

The significance of the sequential processes of phosphate limitation and of phosphate release from medium constituents is demonstrated in technical streptomycin fermentations. The phosphate limitation initiated the streptomycin synthesis as well as the formation of phosphatases and protease. In later periods of the process the phosphate release influences especially the enzyme formation.     

A model of xylitol production by the yeast Candida mogii

Production of xylitol from xylose in batch fermentations of Candida mogii ATCC 18364 is discussed in the presence of glucose as the cosubstrate. Various initial ratios of glucose and xylose concentrations are assessed for their impact on yield and rate of production of xylitol. Supplementation with glucose at the beginning of the fermentation increased the specific growth rate, biomass yield and volumetric productivity of xylitol compared with fermentation that used xylose as the sole carbon source. A mathematical model is developed for eventual use in predicting the product formation rate and yield. The model parameters were estimated from experimental observations, using a genetic algorithm. Batch fermentations, which were carried out with xylose alone and a mixture of xylose and glucose, were used to validate the model. The model fitted well with the experimental data of cell growth, substrate consumption and xylitol production.     

Streptomycin

Summary Streptomyces griseus DTH-2 was grown in 5 1 fermentors on complex media containing calcium carbonate as a buffering agent. It was shown that automatic pH control (4N KOH) could substitute the calcium carbonate giving higher yields of streptomycin. The yield was further increased by omitting inorganic phosphate from the medium and by differential addition of glucose/ammonium sulphate during the fermentation. The maximal yield obtained was 8.5 g of streptomycin per liter.     

Organic acids and penicillin production

Summary Fed-batch fermentations with Penicillium chrysogenum, strain S 3723, were fed with glucose as carbon source or with a mixture of glucose and an organic acid (citrate, lactate, propionate, succinate, formate, or caproate). Increased yields of penicillin-V were observed with, propionate, and formate, 50% higher than in fermentations without acid in the feed. The yield with succinate was improved to the level previously observed with acetate (25%).     

Simultaneous control of ammonium and glucose concentrations in Escherichia coli fermentations

An indirect approach was taken to control the glucose concentration during ammonium-controlled fermentations of Escherichia coli. During ammonium-controlled fermentations, instead of feeding only ammonium, a mixture of glucose and ammonium was fed. The composition of the mixture was estimated from the ratio of glucose to ammonium consumption. With this control system, the ammonium and glucose concentrations were kept quite constant throughout the fermentation. The maximum apparent specific growth rate increased when both ammonium and glucose concentrations were controlled. Utilization of ammonium and glucose, biomass production, and the yields of biomass on both glucose and ammonium all increased when ammonium and glucose were simultaneously controlled.     

Characterization of starch breakdown in the intact spinach chloroplast

Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia oleracea) chloroplast which had been preloaded with ¹⁴C-starch photosynthetically from ¹⁴CO₂. Starch breakdown was dependent upon inorganic phosphate and the ¹⁴C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of ¹⁴C from triose-phosphate to glycerate 3-phosphate. Iodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3-phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was concluded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic cleavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate.     

31P NMR and 13C NMR studies of recombinant Saccharomyces cerevisiae with altered glucose phosphorylation activities

Manipulation of cellular metabolism to maximize the yield and rate of formation of desired products may be achieved through genetic modification. Batch fermentations utilizing glucose as a carbon source were performed for three recombinant strains of Saccharomyces cerevisiae in which the glucose phosphorylation step was altered by mutation and genetic engineering. The host strain (hxk1 hxk2 glk) is unable to grow on glucose or fructose; the three plasmids investigated expressed hexokinase PI, hexokinase PII, or glucokinase, respectively, enabling more rapid glucose and fructose phosphorylation in vivo than that provided by wild-type yeast.Intracellular metabolic state variables were determined by ³¹P NMR measurements of in vivo fermentations under nongrowth conditions for high cell density suspensions. Glucose consumption, ethanol and glycerol production, and polysaccharide formation were determined by ¹³C NMR measurements under the same experimental conditions as used in the ³¹P NMR measurements. The trends observed in ethanol yields for the strains under growth conditions were mimicked in the nongrowth NMR conditions.Only the strain with hexokinase PI had higher rates of glucose consumption and ethanol production in comparison to healthy diploid strains in the literature. The hexokinase PII strain drastically underutilized its glucose-phosphorylating capacity. A regulation difference in the use of magnesium-free ATP for this strain could be a possible explanation. Differences in ATP levels and cytoplasmic pH values among the strains were observed that could not have been foreseen. However, cytoplasmic pH values do not account for the differences observed among in vivo and in vitro glucose phosphorylation activities of the three recombinant strains.     

Protease production by Bacillus subtilis in oxygen-controlled,glucose fed-batch fermentations

Summary An open-loop, on-off control system using the dissolved oxygen level to control a glucose feed was used in a study of growth and production of protease by Bacillus subtilis CNIB 8054. With this system, both glucose and oxygen were controlled at low concentrations. In batch fermentations, protease activity in the fermentation broth was maximum when growth had stopped. During oxygen-controlled, glucose fed-batch fermentations, growth and the production of protease activity continued during glucose feeding. Oxygen-controlled, glucose fed-batch fermentations produced more protease activity than batch fermentations, depending upon the set point for dissolved oxygen. These results indicate that control of glucose and oxygen concentrations can result in improvements in protease production.     

溶剂稳定性蛋白酶产生菌Bacillus licheniformis YP1的发酵优化

溶剂稳定性蛋白酶产生菌Bacillus licheniformis YP1分离自油田土样。考察了碳源、氮源、金属离子等营养因素对YP1菌株发酵产溶剂稳定性蛋白酶的影响。YP1菌株发酵产胞外蛋白酶的最佳碳源为淀粉,果糖、甘露糖和乳糖显著抑制产酶;最佳氮源为酵母膏,干酪素、酵母粉和牛肉膏促进产酶,玉米浆和尿素显著抑制产酶。Mn^2＋可以显著促进酶活,Mg^2＋可以促进产酶,在初步优化的培养条件下,YP1菌株的胞外蛋白酶产量达980U。     

Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium brockii.

Specific changes in the chemical and microbial composition of Thermoanaerobium brockii fermentations were compared and related to alterations of process rates, end product yields, and growth parameters. Fermentation of starch as compared with glucose was associated with significant decreases in growth rate and intracellular fructose-1,6-bisphosphate concentration and with a dramatic increase in the ethanol/lactate product ratio. Glucose or pyruvate fermentation in the presence of acetone was correlated with increased substrate consumption, growth (both rate and yield), acetate yield, and quantitative reduction of acetone to isopropanol in lieu of normal reduced fermentation products (i.e., H2, ethanol, lactate). Acetone altered pyruvate phosphoroclastic activity of cell extracts in that H2, lactate, and ethanol levels decreased, whereas the acetate concentration increased. Glucose fermentation in the presence of exogenous hydrogen was associated with inhibition of endogenous H2 production and either increased ethanol/acetate product ratios and decreased growth at less than 0.5 atm (51 kPa) of H2 or total growth inhibition at 1.0 atm (102 kPA). The effects of exogenous hydrogen on glucose fermentation were totally reversed by the addition of acetone. Glucose fermentation in coculture with Methanobacterium thermoautotrophicum correlated with increased growth (both rate and yield), acetate yield, and the formation of methane in lieu of monoculture reduced products. In coculture, but not monoculture, T. brockii grew on ethanol as the energy source, and acetate and methane were the end products as a direct consequence of hydrogen consumption by the methanogen.     

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae

Bioconversion of xylose—the second most abundant sugar in nature—into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.     

Starch industry wastewater as a substrate for antagonist, Trichoderma viride production

Starch industry wastewater was investigated to assess and improve its potential as a raw material for the conidia production of biocontrol fungi, Trichoderma viride. The wastewater was tested with and without supplements of glucose, soluble starch, meat peptone and probable conidiation inducer chemicals in shake flask culture. Addition of complex carbon source (soluble starch, 1% and 2% w/v) produced maximum conidia ( approximately 3.02 and 4.2 x 10(10)CFU/mL, respectively). On the other hand, glucose addition as a simpler carbon source was either ineffective or, reduced conidia production (from 1.6 x 10(8) in control to 3.0 x 10(7)CFU/mL in 5% w/v glucose supplement). Supplement of nitrogen source showed a small increase of conidia concentration. Propionic, maleic and humic acids, EDTA, pyridine, glycerol and CaCO(3) were examined as probable conidiation inducers and showed effect only on initial rate of conidiation with no increase in final conidia concentration. Intra and extracellular ATP correlation with spore production showed dependence on growth media used and conidia concentration at the end of fermentation. Addition of carbon and nitrogen sources showed an increase in protease activity (from 0.4985 to 2.43 IU/mL) and entomotoxicity (from 10448 to 12335 spruce budworm unit (SBU)/microL). Entomotoxicity was improved by 11% in fermenter over shake flask when starch industry wastewater was supplemented with meat peptone.