Production of indole-3-acetic acid and related indole derivatives from L-tryptophan by <Emphasis Type="Italic">Rubrivivax benzoatilyticus</Emphasis> JA2

Rubrivivax benzoatilyticus JA2 produces indoles with simultaneous utilization of L-tryptophan. Fifteen chromatographically distinct indole derivatives were detected from the L-tryptophan-supplemented cultures of R. benzoatilyticus JA2. Nine of these were identified as, indole 3-acetamide, Methoxyindole-3-aldehyde, indole 3-aldehyde, methoxyindole-3-acetic acid, indole 3-acetic acid, indole-3-carboxylic acid, indole-3-acetonitrile, indole, and trisindoline. Tryptophan stable isotope feeding confirmed the indoles produced are from the supplemented L-tryptophan. Indole 3-acetic acid is one of the major products of L-tryptophan catabolism by R. benzoatilyticus JA2 and its production was influenced by growth conditions. Identification of indole 3-acetamide and tryptophan monooxygenase activity suggests indole 3-acetamide routed IAA biosynthesis in R. benzoatilyticus JA2. The study also indicated the possible multiple pathways of IAA biosynthesis in R. benzoatilyticus JA2.     

l-Phenylalanine catabolism and l-phenyllactic acid production by a phototrophic bacterium,Rubrivivax benzoatilyticus JA2

A phototrophic bacterium (Rubrivivax benzoatilyticus JA2) grows at the expense of l-phenylalanine as sole source of nitrogen but not as carbon source. Near stoichiometric yields of l-phenylpyruvic acid (0.4 mM) and l-phenyllactate (0.4 mM) were observed from l-phenylalanine (0.9 mM consumed). Aminotransfarase and dehydrogenase activities involved in the formation of l-phenylpyruvic acid and l-phenyllactate were demonstrated unequivocally in Rubrivivax benzoatilyticus JA2. Growth conditions and carbon sources had an influence on l-phenyllactate production. The process yielded a maximum of 0.92 mM l-phenyllactate from l-phenylalanine (1 mM) when fructose served as carbon source for R. benzoatilyticus JA2.     

Aniline-Induced Tryptophan Production and Identification of Indole Derivatives from Three Purple Bacteria

Growth on aniline by three purple non-sulfur bacteria (Rhodospirillum rubrum ATCC 11170, Rhodobacter sphaeroides DSM 158, and Rubrivivax benzoatiliticus JA2) as nitrogen, or carbon source could not be demonstrated. However in its presence, production of indole derivatives was observed with all the strains tested. At least 14 chromatographically (HPLC) distinct peaks were observed at the absorption maxima of 275–280 nm from aniline induced cultures. Five major indoles were identified based on HPLC and LC–MS/MS analysis. While tryptophan was the major common metabolite for all the three aniline induced cultures, production of indole-3-acetic acid was observed with Rvi. benzoatilyticus JA2 alone, while indole-3-aldehyde was identified from Rvi. benzoatilyticus JA2 and Rba. sphaeroides DSM 158. Indole-3-ethanol was identified only from Rsp. rubrum ATCC 1170 and anthranilic acid was identified from Rba. sphaeroides DSM 158.     

<Emphasis Type="SmallCaps">l</Emphasis>-Tryptophan catabolism by <Emphasis Type="Italic">Rubrivivax benzoatilyticus</Emphasis> JA2 occurs through indole 3-pyruvic acid pathway

Rubrivivax benzoatilyticus JA2 utilizes l-tryptophan as the sole source of nitrogen for growth, and it has a doubling time of ~11 h (compared to 8 h with ammonium chloride). With cell free extracts in the presence of 2-oxoglutarate, indole-3-pyruvic acid, indole-3-acetaldehyde, indole-3-acetic acid, isatin, benzaldehyde, gallic acid and pyrogallol were identified using high performance liquid chromatography (HPLC) and liquid chromatography–mass spectroscopy (LC–MS) analysis. The conversion of l-tryptophan into indole 3-pyruvic acid and glutamate by an enzyme aminotransferase was confirmed and the catabolism of indole-3-pyruvic acid via side chain oxidation followed by ring oxidation, gallic acid and pyrogallol were confirmed as metabolites. In addition, the proposed pathway sequential conversion of indole-3-pyruvic acid to the end product of pyrogallol was identified, including an enzymatic step that would convert isatin to benzaldehyde by an enzyme yet to be identified. At this stage of the study, the enzyme tryptophan aminotransferase in R. benzoatilyticus JA2 was demonstrated.     

Improvement of 5-aminolevulinic acid production by Rubrivivax benzoatilyticus PS-5 with self-flocculation by co-fermentation of precursors and volatile fatty acids under pH-controlled conditions

Photosynthetic bacteria are known to utilize volatile fatty acids as a carbon source for growth and product formation. In this study, a new isolate, Rubrivivax benzoatilyticus PS-5, possessing self-flocculation properties, was cultivated in modified glutamate-malate (GM) medium containing glutamate and malate as carbon sources. The effect of acetic acid, propionic acid and butyric acid (at 1–4 g L^?1) as co-substrates and 7.5 mM glycine, 10 mM succinic acid as precursors for 5-aminolevulinic acid (ALA) production from R. benzoatilyticus PS-5 was investigated. Among the volatile fatty acids tested, acetic acid was preferred to butyric acid and propionic acid, with the optimum concentrations of 3 g L^?1, 1 g L^?1 and 3 g L^?1, respectively. The highest ALA production was 169.71 μM, 162.16 μM and 46.18 μM, respectively, while the highest productivity was 2.57 μM h^?1, 2.25 μM h^?1 and 0.96 μM h^?1, respectively. The precursor was consumed completely (100 %) while the assimilation of the acetic acid and butyric acid was 62.50 % and 48.65 %, respectively. Supplementation of propionic acid (at 1–4 g l^?1) had a negative effect on growth and ALA production. To increase production efficiency, the pH-control strategy (at pH 6.0–8.0) during fermentation was tested. The optimum pH was 7.0, giving the maximum ALA production of 286.18 μM and a productivity of 3.97 μM h^?1. These values were 1.68-fold and 1.54-fold higher, respectively, than those under uncontrolled pH conditions.     

Evalution of liquid media for fumonisin production byFusarium moniliforme MCR 826

Production of fumonlsins B₁ (FB₁) and B₂ (FB₂) by 5 lyophillzation batches ofFusarium moniliforme strain MRC 826 was studied in several liquid media and vermlculite supplemented with liquid media. In addition the effect of different parameters including pH, Inert material, shake versus stationary cultures as well as different carbon sources on the production of the fumonlsins were investigated. Fumonlsin production in liquid cultures was significantly (P<0.01) correlated (r=0.92–0.98) with fungal growth, which in turn is affected by the pH of the medium as well as the carbon source utilized. The highest FB₁ yields (approximately 40 mg/l) over the incubation period of 14 days were produced in a chemically defined medium with glucose as carbon source set at an initial pH value of 4. FB₁ production in “corn patty” cultures (approximately 1 to 3 g/kg), however, by far exceeded that obtained in the liquid media, while poor fungal growth and fumonlsin production was obtained in vermlculite supplemented cultures. From these studies it became clear that the ability of a culture to produce fumonlsins is determined by the interaction of a variety of physiological and nutritional factors regarding the inoculum and the culture medium.     

Anaerobic degradation of indolic compounds by sulfate-reducing enrichment cultures,and description of Desulfobacterium indolicum gen. nov., sp. nov.

Indole (1.5 mmol/l) added to suflate-rich marine mud or sulfate-free sewage digestor sludge was anaerobically degraded within one week. Enrichments from sludge samples in defined indole-containing media with or without sulfate were selective for sulfate-reducing bacteria or mixed methanogenic associations, respectively. Other enrichments of sulfate-reducing bacteria were obtained with skatole, indoleacetate, indolepropionate, quinoline, and pyridine. From a marine enrichment with indole as sole electron donor and carbon source, an oval to rod-shaped, Gram-negative, nonsporing sulfate-reducing bacterium (strain In04) was isolated. Growth occurred in defined bicarbonate-buffered, sulfide-reduced media supplemented with vitamin B₁₂. Furthen aromatic compounds utilized as electron donors and carbon sources were anthranilic acid and quinoline. Nonaromatic compounds used as substrates were formate, acetate, propionate, ethanol, propanol, butanol, pyruvate, malate, fumarate, and succinate. However, growth with substrates other than indole was rather slow. Thiosulfate served as an alternative electron acceptor. Complete oxidation of indole to CO₂ was shown by stoichiometric measurements in batch culture with sulfate as electron acceptor. An average growth yield of 31.3 g cell dry weight was obtained per mol of indole oxidized. Pigment analysis revealed that cytochromes and menaquinone MK-7 (H₂) were present. Desulfoviridin could not be detected. Strain In04 is described as new species of the new genus Desulfobacterium indolicum.     

Production of Chitinases and β-1,3-Glucanases by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani

The in vitro production of chitinases and β-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO₃. The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for β-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and β-1,3-glucanases were detected. S. elegans culture filtrates, possessing β-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium.     

Studies of the Utilization of Coconut Water Waste for the Production of the Food Yeast Saccharomyces fragilis

The accepted food yeast Saccharomyces fragilis was grown in batch and chemostat culture on coconut water and on a simulated coconut-water medium containing glucose, fructose, sucrose and sorbitol, to provide kinetic data for a feasibility study of microbial protein production. Analyses of growth on individual and mixed carbon substrates were made to determine sugar assimilation patterns in batch and chemostat cultures on coconut water. Growth on the polyol produced a much reduced specific growth rate, assimilation rate, growth yield and productivity compared to growth on the sugars. In mixed substrate fermentations a sequential utilization of the carbohydrates occurred. Both the monosaccharides repressed invertase synthesis and all three sugars repressed sorbitol assimilation. Complete carbon assimilation was only obtained by prolonged batch fermentation or in chemostat cultures at low dilution rates (<0.10 h^-1). Supplementation of coconut water with biotin and nicotinic acid increased biomass yields in chemostat cultures.     

Influence of CO2-HCO3− Levels and pH on Growth, Succinate Production, and Enzyme Activities of Anaerobiospirillum succiniciproducens

Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO₂-HCO₃⁻ growth conditions and suggest that there exists a threshold level of CO₂ for enhanced succinate production in A. succiniciproducens. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO₂-HCO₃⁻ growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K′) was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO₂-HCO₃⁻ as a consequence of regulating electron sink metabolism.     

Impact of carbon sources on growth and oxalate synthesis by the phytopathogenic fungus <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The impact of various supplemental carbon sources (oxalate, glyoxylate, glycolate, pyruvate, formate, malate, acetate, and succinate) on growth and oxalate formation (i.e., oxalogenesis) by Sclerotinia sclerotiorum was studied. With isolates D-E7, 105, W-B10, and Arg-L of S. sclerotiorum, growth in an undefined broth medium (0.1% soytone; pH 5) with 25 mM glucose and 25 mM supplemental carbon source was increased by the addition of malate and succinate. Oxalate accumulation occurred in the presence of glucose and a supplemental carbon source, with malate, acetate, and succinate supporting the most oxalate synthesis. With S. sclerotiorum Arg-L, oxalate-to-biomass ratios, an indicator of oxalogenic potential, were dissimilar when the organism was grown in the presence of different carbon sources. The highest oxalate-to-biomass ratios were observed with pyruvate, formate, malate, acetate, and succinate. Time-course studies with acetate-supplemented cultures revealed that acetate and glucose consumption by S. sclerotiorum D-E7 coincided with oxalogenesis and culture acidification. By day 5 of incubation, oxalogenesis was halted when cultures reached a pH of 3 and were devoid of acetate. In succinate-supplemented cultures, oxalogenesis essentially paralleled glucose and succinate utilization over the 9-day incubation period; during this time period, culture pH declined but never fell below 4. Overall, these results indicate that carbon sources can regulate the accumulation of oxalate, a key pathogenicity determinant for S. sclerotiorum.     

The regulatory effect of fermentable sugar levels on the production of leukotoxin by Actinobacillus actinomycetemcomitans

The relationship between sugar availability and RTX (repeats in toxin) cytotoxin (leukotoxin) production in the periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was investigated using a chemostat. A. actinomycetemcomitans 301-b produced significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures at a dilution rate of 0.15 h⁻¹ and at pH 7.0. When the growth limitation was relieved by pulsing the cultures with 50 or 150 mM fructose (final concentrations), leukotoxin production immediately stopped and the amount of cellular leukotoxin decreased until the culture was returned to fructose-limited conditions. Leukotoxin synthesis was also repressed in the chemostat cultures by pulsing with glucose but not with the non-fermentable sugar analog, α-methyl-d-glucoside. Leukotoxin production was also repressed by fructose in chemostat cultures of ATCC 33384, which is generally recognized as a non-leukotoxin-producing or minimally leukotoxic strain.     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Effect of culture conditions on the production of an extracellular proteinase byThermus sp. Rt41A

Thermus sp. Rt41A produced a single extracellular proteinase, as determined by fast protein liquid chromatography and isoelectric focusing. Proteinase activity was expressed from very early in the log phase, and halted when the growth substrate was exhausted. There was no continued proteinase production in the stationary phase. Proteinase production was not stimulated by O₂ limitation, not repressed by amino acid growth substrates, and its production could not be correlated to the type or oxidation state of the carbon and energy source or the growth rate on different carbon and energy sources. Growth on certain substrates, e.g. glutamate and glucose, resulted in production of high levels of proteinase, whereas others, such as acetate, resulted in low proteinase levels. Acetate repressed proteinase production in cultures growing on L-glutamate. In continuous culture on L-glutamate, acetate or pyruvate, proteinase production was highest at higher growth (dilution) rates. The kinetics of proteinase production in continuous culture on L-glutamate can be interpreted as evidence for the constitutive nature of proteinase expression byThermus sp. Rt41A. The data obtained show that the control of proteinase production is different to that postulated forThermus sp. Ok6.A1.     

Age-dependent and jasmonic acid-induced laticifer-cell differentiation in anther callus cultures of rubber tree

Main conclusion

Callus cultures of rubber tree may serve as an efficient model to screen and study environmental factors and phytohormones that stimulate laticifer cell differentiation and improve latex yield. The number of laticifer cells in bark is one of the most important factors determining the biosynthesis and economic value of rubber trees (Hevea brasiliensis). The differentiation of laticifer cells in planta has been characterized, whereas laticifer-cell differentiation in callus cultures in vitro is largely unknown. In this study, we present molecular and physiological evidences for laticifer-cell differentiation in calli derived from rubber tree anthers. RT-PCR analysis showed that three key genes rubber elongation factor (REF), small rubber particle protein (SRPP), and cis-prenyl transferase (CPT) that are essential in latex biosynthesis in rubber tree bark also were transcribed in anther calli. Laticifer cell development in callus cultures was age-dependent; the cells began to appear at 58 days after initiation of culture, and the percentage of laticifer cells increased steadily with increasing callus age. Addition of 0–2 mg/L jasmonic acid (JA) to the media significantly promoted the differentiation of laticifer cells in callus cultures. However, JA concentrations higher than 3 mg/L were not optimum for laticifer cells differentiation; this result was not observed in previous in planta studies. Laticifer cells differentiated on media with pH 5.8–7.0, with an optimum of pH 6.2, whereas a higher pH inhibited differentiation. These results indicate that the anther-derived rubber tree callus may serve as a new and more efficient model to study environmental factors that influence laticifer cell differentiation, and may be useful for research on new technologies to improve latex yield, and to screen for commercially useful phytohormones.     

A thermostable lipase produced by a newly isolated <Emphasis Type="Italic">Geotrichum</Emphasis>-like strain,R59

Growth and production of lipase by a new Geotrichum-like strain, R59, were studied. Production of extracellular lipase was substantially enhanced when the initial pH of the culture medium, types of carbon and nitrogen sources, substances probably stimulating the lipase biosynthesis, the temperature, and time of growth were optimized. Sucrose and triolein were the most effective carbon sources for lipase production. Maximum lipase activity (146 U/ml^–1) was obtained with urea as the nitrogen source. Growth at 30°C, an initial pH of 6.0 and incubation time of 48 h were found as optimum conditions for cell growth and production of lipase by Geotrichum-like strain R59. The enzyme was thermostable and exhibited very high activity after 1 h incubation at 60°C.     

The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles

The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c.The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles.The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit (aa₃) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4.The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a₃ being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H₂O to OH^? with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.     

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.Abbreviations TDC tryptophan decarboxylase - try L-tyrosine - phe L-phenylalanine - DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate - trp L-tryptophan - E-4-P erythrose-4-phosphate - PEP phosphoenolpyruvate - MDH malate dehydrogenase - G-6-PDH glucose-6-phosphate dehydrogenase - 6-PG-DH 6-phosphogluconate dehydrogenase - Ch-mutase chorismate mutase - AS-synthase anthranilate synthase - n.d. not determined     

Key Process Conditions for Production of C4 Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain

A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter⁻¹ of malate at a yield of 0.42 mol (mol glucose)⁻¹ in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO₃, was required for efficient C₄ dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO₃ dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose)⁻¹, whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO₂, calcium, and O₂). Under optimized conditions, a malate yield of 0.48 ± 0.01 mol (mol glucose)⁻¹ was obtained in bioreactors, a 19% increase over yields in shake flask experiments.In recent years, biologically produced 1,4-dicarboxylic acids (succinate, malate, and fumarate) have attracted great interest as more sustainable replacements for oil-derived commodity chemicals, such as maleic anhydride (50). Malate is currently mainly produced via petrochemical routes for use in food and beverages (18). Development of a biotechnological production process started in the early 1960s with the investigation of the natural malate producer Aspergillus flavus (2). Although process improvements eventually resulted in high product yields and productivities (6), the potential production of aflatoxins (20) prevented the use of this filamentous fungus in industry. Other investigated natural malate-producing fungi (listed in reference 51) produced insufficient malate for industrial use. With the rational design options of metabolic engineering, microorganisms that do not naturally produce large amounts of malic acid may also be considered as production platforms. Wild-type Saccharomyces cerevisiae strains produce little if any malate but would be an interesting starting point for the construction of an efficient malate producer. This yeast has a relatively high tolerance to organic acids and low pH, and due to its role as a model organism in research, a well-developed metabolic engineering toolbox is available. In addition, wild-type S. cerevisiae strains have GRAS (Generally Regarded As Safe) status, so that modified strains are more likely to be allowed in the production of food-grade malic acid.One of the main challenges in the development of an organic acid-producing strain of S. cerevisiae has been the elimination of ethanol formation, which in wild-type strains occurs even under aerobic conditions when glucose concentrations are high (45). Deletion of the pyruvate decarboxylase-encoding genes was found to prevent ethanolic fermentation (17). After evolutionary engineering to remove the growth defects usually associated with pyruvate decarboxylase-negative S. cerevisiae strains, a strain was obtained that produced large amounts of pyruvate, a direct precursor to malate, when grown on glucose (42). Subsequent overexpression of the anaplerotic enzyme pyruvate carboxylase, a cytosolically relocalized malate dehydrogenase and a heterologous malate transporter from Schizosaccharomyces pombe led to a strain that produced significant amounts of malate (51). Cultivation in calcium carbonate (CaCO₃)-buffered shake flasks resulted in malate titers of up to 59 g liter⁻¹ at a yield of 0.42 mol (mol glucose)⁻¹.There are many differences between cultivation in shake flasks and cultivation in (laboratory or industrial) bioreactors. As shake flask cultures lack online pH monitoring and control, there is often significant pH variation over time. The pH is of particular importance. If the yeast can be persuaded to produce organic acids at lower pH values, this reduces the need for active neutralization and thereby reduces by-product formation such as gypsum. However, thermodynamic constraints on acid export, as well as increased stress levels from (undissociated) acid and the low pH, often limit the ability of the microorganisms to produce acids at low pH (32, 43). For this reason, the poorly soluble compound CaCO₃ has traditionally been used to maintain a near-neutral pH in malic acid-producing microbial cultures (6, 29, 51). Adding CaCO₃ also gives increased concentrations of bicarbonate (and thereby CO₂), a substrate for pyruvate carboxylase in the carboxylation of pyruvate (a C₃ carbon molecule) to oxaloacetate (C₄ carbon), as well as calcium. Calcium is known to be involved in cellular signaling pathways (22, 26, 33, 46) and to influence pyruvate carboxylase activity (21, 24). Finally, oxygen transfer rates in shake flasks are often poor compared to those in stirred (laboratory) bioreactors. The formation of significant concentrations (25 g liter⁻¹) of glycerol, a well-known redox sink in S. cerevisiae (41), in shake flask cultures of the engineered malate-producing strain (51) was a strong indication of oxygen limitation.Initial experiments in aerobic, pH-controlled bioreactor cultures of the malate- and succinate-producing Saccharomyces cerevisiae strain RWB525 yielded only low concentrations of these C₄ dicarboxylic acids. The goal of the present study was to identify process parameters that explain the different production levels in shake flask and bioreactor cultures. To this end, we analyzed, both separately and in combination, the impact of culture pH and concentrations of calcium, carbon dioxide, and oxygen on the production of malate and succinate.     

Growth conditions of clostridium perfringens type B for production of toxins used to obtain veterinary vaccines

The diseases caused for Clostridium perfringens are generically called enterotoxemias because toxins produced in the intestine may be absorbed into the general circulation. C. perfringens type B, grown in batch fermentation, produced toxins used to obtain veterinary vaccines. Glucose in concentrations of 1.4–111.1 mM was used to define the culture medium. The minimum concentration for a satisfactory production of vaccines against clostridial diseases was 55.6 mM. Best results were brought forth by meat and casein peptones, both in the concentration 5.0 g l^?1 in combination with glucose and a culture pH maintained at 6.5 throughout the fermentation process. The production of lactic, acetic and propionic organic acids was observed. Ethanol was the metabolite produced in the highest concentration when cultures maintained steady pH of 6.5 with exception of cultures with initial glucose concentration of 1.4 mM, where the highest production was of propionic acid. Maximal cell concentration and the highest toxin title concomitantly low yield coefficient to organic acids and ethanol were obtained using basal medium containing 111.1 mM glucose under a controlled pH culture (pH) 6.5 in batch fermentations of C. perfringens type B. These data contribute to improve process for industrial toxin production allowing better condition to produce a toxoid vaccine.