Production of Propionibacterium shermanii biomass and vitamin B12 on spent media

AIMS: The propionibacteria are commercially important due to their use in the cheese industry, and there is a growing interest for their probiotic effects. Stimulatory effects of lactic acid bacteria (LAB) on propionic acid bacteria have been observed. This study was designed to examine the possibility of using spent media previously used to grow LAB for the production of biomass and metabolites of Propionibacterium freudenreichii subsp. shermanii. METHODS AND RESULTS: Seventeen MRS and vegetable juice media were prefermented by various LAB and evaluated for their ability to subsequently support the growth of Propionibacterium, using automated spectrophotometry (AS). Growth of Propionibacterium in spent media was strongly affected by the LAB strain used to produce the spent medium. The native MRS medium (not prefermented) yielded the highest optical density values followed by prefermented media by Lactobacillus acidophilus, Bifidobacterium longum and Lactococcus lactis. Prefermented cabbage juice enabled good growth of Propionibacterium. For the production of organic acids and vitamin B12, cells of Propionibacterium were concentrated and immobilized in alginate beads in the aim of accelerating the bioconversions. More propionic acid was obtained in spent media than in native MRS. The concentration of vitamin B12 was higher in media fermented with free cells than those with immobilized cultures; with the free cells, its concentration varied from 900 to 1800 ng ml(-1) of media. CONCLUSIONS: It was demonstrated that spent media could be recycled for the production of Propionibacterium and metabolites, depending on the LAB strain that was previously grown. Media remediation is needed to improve the production of vitamin B12, especially with immobilized cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents an option for recycling of spent media generated by producers of LAB or producers of fermented vegetables. The propionic fermentation may result in three commercial products: biomass, vitamin B12 or organic acids, which may be used as starters, supplements or food preservatives. It is an attractive process from economical and environmental standpoints.     

Growth engineering of Propionibacterium freudenreichii shermanii for organic acids and other value-added products formation

Propionic acid production from glucose was studied using Propionibacterium freudenreichii shermanii. Conditions were optimized for high yields of propionic acid and total organic acids by sequential optimization of parameters like pH, inoculum age, inoculum volume and substrate concentration. Near-theoretical yield (0.54?±?0.023?g/g) was achieved for propionic acid with fermentation of 1% glucose using 20% (v/v) of 48?hr old P. shermanii at 30°C, pH maintained at 5.5. Total organic acid yield under these conditions was 0.74?±?0.06?g/g. The study resulted in achieving 98% and 95% theoretical yields of propionic acid and total organic acids, respectively. Under optimized conditions, along with organic acids, P. shermanii also produced vitamin B12 and trehalose intracellularly, showing its potential to be used as a cell factory.     

Utilization of different carbon sources by bacteria isolated from the roots of pine seedlings (Pinus silvestris L.) inoculated with root-free, rhizosphere and mycorrhizosphere soil

Studies were carried out on the utilization of different sugars (glucose, fructose, arabinose, xylose, sucrose) and organic acids (acetic, citric, fumaric, propionic, succinic) by fast and slow growing bacteria isolated from the roots of pine seedlings (P. silvestris L.) inoculated with root-free, rhizosphere and mycorrhizosphere soil from nursery and mature pine forest. Sucrose among sugars and propionic acid among organic acids were the less frequently utilized compounds. Sugars were better carbon sources than organic acids. Proportion of isolates utilizing respective sugars or organic acids was, in general, significantly higher among fast growing bacteria as compared with slow growing ones. No significant differences in number of strains assimilating the appropiate sugars depending on their original habitat or kind of soil were observed. Such differences were noted for utilization of some organic acids. 3-factor ANOVA confirmed that the growth speed of bacteria at the moment of their isolation had the strongest effect on utilization of the compounds studied.     

Effect of Organic Acids on Shrimp Pathogen, <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

Shrimp farming accounts for more than 40% of the world shrimp production. Luminous vibriosis is a shrimp disease that causes major economic losses in the shrimp industry as a result of massive shrimp kills due to infection. Some farms in the South Asia use antibiotics to control Vibrio harveyi, a responsible pathogen for luminous vibriosis. However, the antibiotic-resistant strain was found recently in many shrimp farms, which makes it necessary to develop alternative pathogen control methods. Short-chain fatty acids are metabolic products of organisms, and they have been used as food preservatives for a long time. Organic acids are also commonly added in feeds in animal husbandry, but not in aquaculture. In this study, growth inhibitory effects of short-chain fatty acids, namely formic acid, acetic acid, propionic acid, and butyric acid, on V. harveyi were investigated. Among four acids, formic acid showed the strongest inhibitory effect followed by acetic acid, propionic acid, and butyric acid. The minimum inhibitory concentration (MIC) of 0.035% formic acid suppressed growth of V. harveyi. The major inhibitory mechanism seems to be the pH effect of organic acids. The effective concentration 50 (EC₅₀) values at 96 h inoculation for all organic acids were determined to be 0.023, 0.041, 0.03, and 0.066% for formic, acetic, propionic, and butyric acid, respectively. The laboratory study results are encouraging to formulate shrimp feeds with organic acids to control vibrio infection in shrimp aquaculture farms.     

Characterization of anaerobic microbial culture with high acidogenic activity

The mixed cultures which were used were isolated from municipal sludge digesters, and the production of organic acids (acetic, propionic, butyric, etc.) from carbohydrates was tested. The behavior of the reference population (culture R) obtained directly from the sewage treatment plant, is compared to that obtained after three months in a plug-flow reactor (Gradostat fermentor) without pH control (culture A) and after six months with pH control (culture B). For culture B, the specific rate of acid production is related to the cell growth rate by (1/X)r_p= 17 µ + 1.6 with a maximal acid concentration of 40 g/liter. The batch culture yields are improved from 0.36g/g for the initial culture (R) to 0.72 g/g for culture B after six months in continuous culture, and 0.8 g/g in plug-flow continuous culture. The productivity of organic acids reaches 1.7 g/liter·hr. It is suggested that the acidogenic fermentation, the first step of methanogenesis, is a potential process to produce acetic, propionic, and butyric acids.     

Acetoin production as an indicator of growth and metabolic inhibition of Listeria monocytogenes

It has been shown that Listeria monocytogenes produces acetoin from glucose under aerobic conditions. A defined medium with glucose as the sole carbon source was used in an aerobic shake flask culture to reliably produce acetoin. Acetoin, the reactive compound in the Voges-Proskauer test, was assayable in the medium and was used to quantify the metabolic response when inhibitors were added to the medium. Inhibitors such as lactic, acetic, propionic and benzoic acids were used to demonstrate the utility of acetoin production as an indicator of metabolic disruption. With increasing levels of inhibitor, the metabolic and growth responses were measured by acetoin production and optical density change, respectively. Both measurements decreased in a similar manner with increasing inhibitor concentrations. The data also showed the apparent mode of action of the inhibitors. A bacteriostatic effect was observed for the protonated organic acids, acetic (4 mmol l(-1)) and propionic (4 mmol l(-1)), whereas protonated lactic (4 mmol l(-1)) and benzoic (0.16 mmol l(-1)) acids gave an irreversible (apparent bacteriocidal) effect. Lactic, acetic, and propionic acids showed stimulation of metabolic activity at low concentrations, but benzoic did not. Acetoin production is a novel method for quantifying and assessing the mode of action of inhibitors against L. monocytogenes. This system can be used to screen inhibitors for applications in food safety.     

The effect of propionic to acetic acid ratio on anaerobic-aerobic (low dissolved oxygen) biological phosphorus and nitrogen removal

In this paper, three lab-scale sequencing batch reactors (SBR-A, B, and C) operated with anaerobic/aerobic (low dissolved oxygen, 0.15-0.45 mg L(-1)) configuration were long-term cultured, respectively with single acetic acid and propionic/acetic acid of 1/1 and 2/1 (carbon molar ratio), and the comparisons of anaerobic and aerobic transformations of phosphorus and nitrogen among them were made. With the increase of propionic/acetic acid, lower anaerobic phosphorus release and higher phosphorus release to short-chain fatty acids uptake ratio were observed, and less anaerobic and aerobic transformations of glycogen and poly-3-hydroxybutyrate as well as total polyhydroxyalkanoates occurred, but the transformations of poly-3-hydroxyvalerate and poly-3-hydroxy-2-methyvalerate increased. The phosphorus removal efficiency was respectively 81, 94 and 97% in SBR-A, B and C. Almost all ammonium was removed and no significant nitrite was accumulated at different propionic/acetic acid ratios. However, the nitrate accumulation and total nitrogen removal were observed to be affected by propionic/acetic acid ratio. The total nitrogen removal efficiency was 61, 68 and 82%, and the aerobic end nitrate concentration was 8.05, 6.40 and 3.54 mg L(-1) in three SBRs, respectively. All the above studies indicated that the sole acetic acid caused more nitrate accumulation than propionic and acetic acids mixture, and a pertinent increase of wastewater propionic/acetic acid ratio was of benefit to both nitrogen and phosphorus removal in an anaerobic/aerobic (low dissolved oxygen) biological wastewater treatment process.     

Listeria contamination at a poultry processing plant

Escherichia coli grown in broth initially at pH 5.0 (pH 5.0-grown organisms) survived exposure to inorganic acid or to acid pH plus organic acid which prevented subsequent growth by pH 7.0-grown organisms. This resistance of pH 5.0-grown organisms to organic acids was observed at acid pH with lactic, propionic, benzoic, sorbic, trans-cinnamic and acetic acids. Such resistance might allow acid-habituated organisms to survive in acid foods or at body sites such as the urinary tract where organic acids are present at acid pH.     

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.     

Effect of organic acid on gastrointertinal motility of rat invitro

The quantity of organic acids ( lactic acid, acetic acid, propionic acid and butyric acid ) in the content of the gastrointestinal tract of germ-free and conventional rats and the $\text{in}$$\text{vitro}$ effects of the organic acid on the motility of the gastrointestinal tract of rats were investigated.Organic acids were detected only in the gastrointestinal contents of conventional rats but not in those of germ-free rats.Lactic acid detected in the stomach of rats stimulated the motility of both small and large bowel while acetic acid, propionic acid and butyric acid found in the cecum stimulated the motility of the large bowel but not of small bowel.     

Synthesis of organic acids by immobilized propionic bacteria in the flow system and stabilization of the process

The capacity of immobilized cells of propionic bacteria to synthesize organic acids was examined. Propionibacterium shermanii cells incorporated into polyacrylamide gel were capable to synthesize propionic, acetic and pyruvic acids in the flow system. As a carbon source glucose, lactate-Na or whey lactose was used. The greatest amount of the acids was synthesized with the use of lactate-Na. The life-time of the biocatalyst (immobilized cells) can be increased by its reactivation with a nutrient medium required for optimal cell proliferation.     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

Effect of organic acids on the biosynthesis of macrotetralide antibiotics by an Actinomyces chrysomallus var. carotenoides strain

The biosynthesis of macrotetrolides by Actinomyces chrysomalus var. carotenoides was stimulated by acetic, succinic, propionic, oxalic, malic, tartaric, citric, pyruvic, alpha-ketoglutaric and fumaric acids. Incorporation of 14C-acetate into the molecule of the antibiotic and the data on dependence of the stimulating effect upon the quantitative ratio and time of the organic acid addition were indicative of the role of acetic, succinic and propionic acids as precursors of macrotetrolides. The other organic acids increased the biosynthesis of macrotetolides when added to the culture within wide time ranges of the culture development and prolonged the period of the mycelium productive state.     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

Acetoin production as an indicator of growth and metabolic inhibition of Listeria monocytogenes

It has been shown that Listeria monocytogenes produces acetoin from glucose under aerobic conditions. A defined medium with glucose as the sole carbon source was used in an aerobic shake flask culture to reliably produce acetoin. Acetoin, the reactive compound in the Voges–Proskauer test, was assayable in the medium and was used to quantify the metabolic response when inhibitors were added to the medium. Inhibitors such as lactic, acetic, propionic and benzoic acids were used to demonstrate the utility of acetoin production as an indicator of metabolic disruption. With increasing levels of inhibitor, the metabolic and growth responses were measured by acetoin production and optical density change, respectively. Both measurements decreased in a similar manner with increasing inhibitor concentrations. The data also showed the apparent mode of action of the inhibitors. A bacteriostatic effect was observed for the protonated organic acids, acetic (4 mmol l⁻¹) and propionic (4 mmol l⁻¹), whereas protonated lactic (4 mmol l⁻¹) and benzoic (0·16 mmol l⁻¹) acids gave an irreversible (apparent bacteriocidal) effect. Lactic, acetic, and propionic acids showed stimulation of metabolic activity at low concentrations, but benzoic did not. Acetoin production is a novel method for quantifying and assessing the mode of action of inhibitors against L. monocytogenes . This system can be used to screen inhibitors for applications in food safety.     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

Changes in Membrane Lipids of Roots Associated with Changes in Permeability: I. EFFECTS OF UNDISSOCIATED ORGANIC ACIDS

Previous work has shown that undissociated forms of organic acids, such as formic, acetic, and propionic acids, increase the permeability of barley roots to ions. The work here was undertaken to test whether these undissociated acids affect the lipids from the root membranes in such a way as to account for the permeability increase. Relative amounts of the principal fatty acids from barley root membranes were measured as a function of organic acid concentration, pH, and time of treatment of barley roots under conditions similar to those of the previous studies.     

Effect in the cow of intraruminal infusions of volatile fatty acids and of lactic acid on the secretion of the component fatty acids of the milk fat and on the composition of blood

1. The effects in the cow of intraruminal infusions of acetic acid, propionic acid or butyric acid on the secretion of the component fatty acids of the milk fat, and of these acids and of lactic acid on the composition of the blood plasma of the jugular vein, have been studied. 2. The infusion of acetic acid or butyric acid increased the yield of the C₄–C₁₆ acids of milk fat but decreased the yield of C₁₈ acids. The infusion of propionic acid decreased the yields of all major component acids except palmitic acid and possibly lauric acid. 3. The changes in the concentrations in blood plasma of glucose and of ketone bodies were consistent with the glucogenic effect of propionic acid and the ketogenic effects of butyric acid and acetic acid. The effects of lactic acid were not consistent from cow to cow. Only with the infusion of acetic acid was a significant increase in the concentration of total volatile fatty acids in blood plasma found. Infusions of butyric acid and of propionic acid tended to depress the concentration of citric acid in the blood plasma and infusion of acetic acid increased it. No consistent effects of the infused acids on the concentration in blood plasma of esterified cholesterol, free cholesterol, triglyceride or phospholipid were observed. 4. The possibility is discussed that the effects of the infused acids on milk-fat secretion are caused through an alteration of the concentrations of precursors of milk fat in mammary arterial blood.     

Effect of short-chain acids on the carboxymethylcellulase activity of the ruminal bacterium<Emphasis Type="Italic">Ruminococcus albus</Emphasis>

The addition of 100-300 mmol/L of acetic, propionic, butyric or lactic acids (short-chain acids), or of acetic, propionic, and butyric acids (volatile fatty acids, VFA) mixtures increased the degradation of carboxymethyl cellulose (CMC) by R. albus (7.5 to 46 and 6 to 39 %, respectively). Differences among individual acids were observed at 300 mmol/L whereas VFA mixtures differed at 100 mmol/L. When assayed at the same concentration, CMCase activity was increased less by NaCl than by the short-chain acids, whereas ethylene glycol decreased the activity. Since osmolarity and/or ionic strength changes in the medium cannot completely account for the observed increases of carboxymethylcellulase (CMCase) activity, it is suggested that the anions of short-chain acids produce changes in the reaction media polarity that contribute to the effects observed. Alterations in the media could also bring about conformational changes in CMCase leading to increased rates of reaction and subsequent increases in CMC degradation. Finally, explanations for the observed phenomena based on the direct effect of the compounds tested on the cellulosome complex, its domains, and/or its component enzymes are proposed.