Production of propionic acid by mixed cultures ofPropionibacterium shermanii andLactobacillus casei in autoclave-sterilized whey

Summary Pure cultures ofPropionibacterium freudenreichii ss.shermanii did not grow in autoclave-sterilized cheese whey (121°C, 15 psi, 20 min) at whey concentrations greater than 2% (w/v) spray-dried sweet dairy whey. Propionic acid was produced from autoclave-sterilized whey by growingP. shermanii in mixed culture withLactobacillus casei. In medium containing 5–12% autoclaved whey solids and 1% yeast extract, the mixed culture produced 1.3–3.0% propionic acid, 0.5–1.0% acetic acid, and 0.05–0.80% lactic acid. All the lactose was consumed. Using pH-controlled fermentors (pH=7.0), mixed cultures produced at least 30% more propionic acid than cultures in which pH was not controlled.     

Methane production from organic acid-rich wastewaters by immobilized thermophilic methane-producing bacteria

Thermophilic methane-producing bacteria isolated from a wastewater treatment facility have been immobilized in acetylcellulose filter with agar. The immobilized cells produced methane from wastewaters in rich organic acid (acetic, propionic and butyric acids) at the rate of 1.4 μmol mg protein⁻¹ h⁻¹. The optimum conditions for methane production by immobilized whole cells were 52–55°C and pH 7.0–8.0. The immobilized cells retained 80% of the initial activity after exposure to air. The immobilized thermophilic bacteria produced methane continuously over 10 days at 52°C.     

Accumulation of propionic acid during anaerobic treatment of distillery wastewater from barley-Shochu making

Synthetic wastewater consisting aliphatic acids contained in distillery wastewater from barley-shochu making was treated anaerobically. It was suggested that propionic acid was produced from lactic acid and citric acid via succinic acid. Since it appears to be difficult to treat anaerobically wastewater in which propionic acid is accumulated, we attempted to repress the production of propionic acid during acidification. The amount of propionic acid produced increased with an increase in the hydraulic retention time (HRT) at pH 7. Although the treatment was examined using different pHs at a shorter HRT of 10 h, it was difficult to repress the production of propionic acid.     

Propionic acid fermentation of lactose by Propionibacterium acidipropionici: effects of pH

Batch propionic acid fermentation of lactose by Propionibacterium acidipropionici were studied at various pH values ranging from 4.5 to 7.12. The optimum pH range for cell growth was between 6.0 and 7.1, where the specific growth rate was approximately 0.23 h(-1). The specific growth rate decreased with the pH in the acids have been identified as the two major fermentation products from lactose. The production of propionic acid was both growth and nongrowth associated, while acetic acid formation was closely associated with cell growth. The propionic acid yield increased with decreasing pH; It changed from approximately 33% (w/w) at pH 6.1-7.1 to approximately 63% at pH 4.5-5.0. In contrast, the acetic acid yield was not significantly affected by the pH; it remained within the range of 9%-12% at all pH values. Significant amounts of succinic and pyruvic acids were also formed during propionic acid fermentation of lactose. However, pyruvic acid was reconsumed and disappeared toward the end of the fermentation. The succinic acid yield generally decreased with the pH, from a high value of 17% at pH 7.0 to a low 8% at pH 5.0 Effects of growth nutrients present in yeast ex-tract on the fermentation were also studied. In general, the same trend of pH effects was found for fermentations with media containing 5 to 10 g/L yeast extract. However, More growth nutrients would be required for fermentations to be carried out efficienytly at acidic pH levels.     

Short-chain organic acids at pH 5.0 kill Escherichia coli and Salmonella spp. without causing membrane perturbation

When strains of Escherichia coli K12 and Salmonella spp. were incubated with 0.5–0.7 mol/l formic or propionic acid at pH 5.0, propionic acid was more active than formic acid. It killed 90% of the cell population within 60 min compared with over 3 h for formic acid. Cell death was not associated with a reduction in culture turbidity or a loss of membrane integrity since morphologically normal membranes were observed by electron microscopy and only a small proportion of the cytoplasmic enzyme β-galactosidase leaked into the supernatant fluid of acid-treated E. coli K12 cultures.     

Short-chain organic acids at ph 5.0 kill Escherichia coli and Salmonella spp. without causing membrane perturbation

When strains of Escherichia coli K12 and Salmonella spp. were incubated with 0.5-0.7 mol/l formic or propionic acid at pH 5.0, propionic acid was more active than formic acid. It killed 90% of the cell population within 60 min compared with over 3 h for formic acid. Cell death was not associated with a reduction in culture turbidity or a loss of membrane integrity since morphologically normal membranes were observed by electron microscopy and only a small proportion of the cytoplasmic enzyme beta-galactosidase leaked into the supernatant fluid of acid-treated E. coli K12 cultures.     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

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.     

Propionic acid fermentation of ultra-high-temperature sterilized whey using mono-and mixed-cultures

Summary Unlike sterilization by autoclave (Anderson et al. 1986) high concentrations of cheese whey sterilized by ultra high temperature (UHT) resulted in a medium conducive to microbial growth and propionic acid production. Propionibacterium freudenreichii ss. shermanii, grown with pH control in 12% whey solids and 1% yeast extract sterilized by UHT, produced about 1.9% propionic acid within 70 h; more than 50% of the lactose was not used. Under similar conditions, mixed cultures of P. shermanii and Lactobacillus casei produced more than 3.0% propionic acid. Acclimating the mixed culture to the whey medium resulted in 4.5% propionic acid. The amount of propionic acid produced was further increased to about 6.5% by raising the concentration of whey solids to about 18%. Using the mixed culture, all the lactose was consumed and lactic acid did not accumulate.     

Physiological criteria and mycotoxin production as AIDS in identification of common asymmetric penicillia

The taxonomy of the asymmetric (predominantly terverticillate) penicillia is based on morphological differences that leave identification difficult. The application of physiological criteria facilitated the identification of the common asymmetric penicillia investigated. Changes in the placement of some strains of these penicillia made the connection to mycotoxin-producing ability clearer. The classical criterion of conidium color was deemphasized and replaced by the following criteria: (i) growth on nitrite-sucrose agar and (ii) growth and acid (and subsequent base) production on creatine-sucrose agar (containing bromocresol purple). Other criteria used or developed were: (iii) growth on sorbic acid plus benzoic acid agar (50 + 50 ppm, pH 3.8), (iv) growth on an agar containing 1,000 ppm propionic acid (pH 3.8), (v) growth on an agar containing 0.5% acetic acid, (vi) growth at 37 degrees C, (vii) growth rate on an agar containing 0.1% pentachloronitrobenzene, (viii) production of extracellular tricaproinase, and (ix) fasciculation on a medium containing 10 ppm botran (2,6-dichloro-4-nitroanilin). The pattern of extracellular metabolites after thin-layer chromatography was used as a chemotaxonomic criterion. The species investigated, the number of isolates investigated, and the toxins which some of these isolates produce were: Penicillium roqueforti (18) (patulin), P. citrinum (11) (citrinin), P. patulum (9) (patulin and griseofulvin), P. expansum (patulin and citrinin), P. hirsutum (13), P. brevicompactum (19), and P. chrysogenum (12). Widespread species of the P. cyclopium, P. viridicatum, and P. expansum series of Raper and Thom (A Manual of the Penicillia, 1949) were subdivided into four new groups: "P. crustosum pA" (29) (penitrem A), "P. melanochlorum" (29), "P. cyclopium p" (119) (penicillic acid and infrequently penitrem A), and "P. viridicatum o-c" (43) (ochratoxin A and citrinin). "P. viridicatum o-c" was separated from "P. cyclopium p" due to its ability to grow on nitrite as sole nitrogen source. The species and groups investigated were related to the new taxonomic classification of the genus Penicillium according to Pitt.     

Effect of pH on organic acid production byClostridium propionicum in test tube and fermentor cultures

Summary Clostridum propionicum is a chemical autotroph that metabolizes alanine to propionic acid (reduction product) and acetic acid (oxidation product). The ratio of propionate/acetate predicted by the electron balance is 2:1. This study reports the effect of pH on growth and organic acid production by this organism when grown in both test tube cultures initially buffered from pH 7.0 to 5.0, and in fermentors maintained at pH 7.0 and 6.5. Highest growth and organic acid production was found at pH 7.0 in both cases. HPLC analysis showed that at pH 7.0, the ratios of propionate to acetate were 0.45:1 (stationary tube, 24 h). The highest ratio observed was 1.8:1 (stationary tube, pH 6.0, 24h). This tube produced 8.5% of the acids produced in the pH 7.0 culture tube. The identify of the major portion of the reduction products of the organism remains unknown.     

Effects of potassium sorbate and other antibotulinal agents on germination and outgrowth of Clostridium botulinum type E spores in microcultures.

The effects of potassium sorbate, sodium hypophosphite, sodium tripolyphosphate, sodium nitrite, and linoleic acid on the germination and outgrowth of Clostridium botulinum type E spores were studied in microcultures. At pH 5.8 to 6.0 in liver veal agar, the germination rate was decreased to nearly zero with 1.0, 1.5, or 2.0% sorbate. At pH 7.0 t 7.2, these levels of sorbate afforded germination and outgrowth of abnormally shaped cells that were defective in cell division. At the high pH range, 0.5 or 1.0% hypophosphite had effects similar to those of sorbate. The use of 0.05% sodium nitrite with sorbate enhanced the lysis of outgrowing cells at pH 7.2 or lower. Emergence and elongation were inhibited by 0.05% linoleic acid with or without 1.0% sorbate at pH 7.0 to 7.2. The addition of 0.5% tripolyphosphate to media containing 1.5% sorbate at pH 7.1 prevented normal cell growth to an extent greater than with sorbate alone.     

Influence of pH on organic acid production by Clostridium sporogenes in test tube and fermentor cultures.

The influence of pH on the growth parameters of and the organic acids produced by Clostridium sporogenes 3121 cultured in test tubes and fermentors at 35 degrees C was examined. Specific growth rates in the fermentor maintained at a constant pH ranged from 0.20 h-1 at pH 5.00 to 0.86 h-1 at pH 6.50. Acetic acid was the primary organic acid in supernatants of 24-h cultures; total organic acid levels were 2.0 to 22.0 mumol/ml. Supernatants from pH 5.00 and 5.50 cultures had total organic acid levels less than one-third of those found at pH 6.00 to 7.00. The specific growth rates of the test tube cultures ranged from 0.51 h-1 at pH 5.00 to 0.95 h-1 at pH 6.50. The pH of the medium did not affect the average total organic acid content (51.5 mumol/ml) but did affect the distribution of the organic acids, which included formic, acetic, propionic, butyric, 3-(p-hydroxyphenyl)propionic, and 3-phenylpropionic acids. Butyric acid levels were lower, but formic and propionic acid levels were higher, at pH 5.00 than at other pHs.     

Influence of pH on organic acid production by Clostridium sporogenes in test tube and fermentor cultures

The influence of pH on the growth parameters of and the organic acids produced by Clostridium sporogenes 3121 cultured in test tubes and fermentors at 35 degrees C was examined. Specific growth rates in the fermentor maintained at a constant pH ranged from 0.20 h-1 at pH 5.00 to 0.86 h-1 at pH 6.50. Acetic acid was the primary organic acid in supernatants of 24-h cultures; total organic acid levels were 2.0 to 22.0 mumol/ml. Supernatants from pH 5.00 and 5.50 cultures had total organic acid levels less than one-third of those found at pH 6.00 to 7.00. The specific growth rates of the test tube cultures ranged from 0.51 h-1 at pH 5.00 to 0.95 h-1 at pH 6.50. The pH of the medium did not affect the average total organic acid content (51.5 mumol/ml) but did affect the distribution of the organic acids, which included formic, acetic, propionic, butyric, 3-(p-hydroxyphenyl)propionic, and 3-phenylpropionic acids. Butyric acid levels were lower, but formic and propionic acid levels were higher, at pH 5.00 than at other pHs.     

Bioconversion of benzonitrile to benzoic acid using free and agar entrapped cells of Nocardia globerula NHB-2

The free and agar immobilized cells of Nocardia globerula NHB-2 having nitrilase (EC 3.5.5.1) activity were used to catalyse the transformation of benzonitrile to benzoic acid. The whole cells of N. globerula NHB-2 were immobilized in agar which exhibited maximum conversion of benzonitrile to benzoic acid in 0.1 M potassium phosphate buffer pH 7.5 (free cells) 8.0 (immobilized cells), temperature 40 degrees C, cells 2 mg dcm ml(-1) reaction mixture and benzonitrile (4% v/v) in 4 h (free cells). The effect of temperature on the stability of nitrilase was studied and cells retained 100% activity at 30 degrees C and lost 50% activity at 40 degrees C. In a fed batch mode of reaction 108 and 84 gl(-1) benzoic acid was produced using free and agar entrapped cells (2 g dcm). The agar immobilized cells were recycled up to three times and 80, 62, 20 gl(-1) benzoic acid was again produced respectively in each of three cycles and a total 244 g benzoic acid was produced by recycling the same mass of immobilized biocatalyst.     

Anaerobic acidogenesis of a complex wastewater: I. The influence of operational parameters on reactor performance

The influence of operational, parameters, such as hydraulic retention time, organic loading rate, influent substrate concentration, pH, and temperature, on the performance of the first phase of anaerobic digestion has been investigated. A complex substrate based on beef extract was used, and six series of experimental runs were conducted, each one showing the effect of one operational variable. The predominant fermentation products were always acetic and propionic acid, independent of the values of the operational parameters. For initial COD concentrations and hydraulic retention times above the critical values identified as 3 g/L and 6 h, respectively, the degree of acidification achieved was between 30 and 60%. The degree of acidification was found to increase with the hydraulic retention time and decrease with the influent substrate concentration and organic loading rate, while the opposite held true for the rate of product formation. Furthermore, it has been demonstrated that acidification is primarily determined by the hydraulic retention time and the rate of product formation by the influent substrate concentration. The concentration of the acetic acid produced was found to depend on the operational parameters. However, the concentration of propionic acid produced depended only on the substrate availability with a consistent proportion of 8% initial COD converted to it. The optimum pH and temperature were 7 and 40 degrees C, respectively. The percentage of acetic acid as a proportion of the total volatile fatty acids produced was found to increase with increasing pH and temperature, while the percentage of propionic acid seemed to decrease accordingly. Finally the effect of the temperature on the rate of acidification followed an Arrhenius type equation with an activation energy equal to 4739 cal/mol.     

Effect of propionic acid on poly (β-hydroxybutyric-co-β-hydroxyvaleric) acid production byAlcaligenes eutrophus

Summary The effect of propionic acid on poly(-hydroxybutyric-co--hydroxyvaleric)acid P(HB-co-HV) copolymer production byAlcaligenes eutrophus ATCC 17699 supplied with fructose and propionic acid under nitrogen limited conditions was studied. The growth ofA. eutrophus was almost completely inhibited when the concentraion, of propionic acid exceeds 1.5 g/L. Specific production rate of HV unit was highest when propionic acid concentration was 0.5 g/L. In batch culture, pH change occurs in proportion to the consumption of propionic acid. Optimal concentration of propionic acid was maintained during the production phase by using a pH-stat feeding method and a total polymer content higher than 70% and the relative HV content upto 50% could be achieved.     

Effect of solids retention time and temperature on waste activated sludge hydrolysis and short-chain fatty acids accumulation under alkaline conditions in continuous-flow reactors

The effects of solids retention time (SRT) and temperature on waste activated sludge (WAS) hydrolysis and short-chain fatty acids (SCFAs) accumulation were investigated in a series of continuous-flow reactors at pH 10. The experimental results showed that the increase of either SRT or temperature benefited the hydrolysis of WAS and the production of SCFAs. The changes in SRT gave also impact on the percentage of acetic and propionic acids in the fermentative SCFAs, but little influence on that of the slightly long-chain SCFAs, such as n-butyric, iso-butyric, n-valeric and iso-valeric acids. Compared with the control (pH unadjusted) experiment, at SRT of 12d and temperature of 20 degrees C the concentration of SCFAs produced at pH 10 increased from 261.2 to 933.5mg COD/L, and the propionic acid percentage improved from 11.7 to 16.0%. It can be concluded from this investigation that the efficient continuous production of SCFAs at pH 10 is feasible.     

Characterization of a dextranolytic biotype of Flavobacterium multivorum from soil

Bacteria obtained by enrichment on dextran as sole carbon source before plating, or by direct plating of soil suspensions on dextran agar, included a yellow, non-motile, Gram negative rod with distinctive morphology and ultrastructure in negatively stained preparations under the electron microscope. The 16 isolates obtained all showed asymmetric division. In a small proportion of the population a phenomenon resembling budding resulted in the release of spherical cells. The isolates were compared with Flavobacterium breve , in which a similar morphology has been observed, but were clearly distinct on physiological grounds. The collection of strains formed a moderately uniform phenotype similar to, but generally more active biochemically, than F. multivorum ; all produced acid from dextran and grew on dextran as sole carbon source. Seven of the isolates produced pits in pectate gel at neutral and alkaline pH, but none broke down cellulose or chitin. Flavobacterium multivorum which was originally described from human clinical sources can be readily isolated from soil by the dextran enrichment technique.     

Effect of short-chain organic acids on macromolecular synthesis in Escherichia coli

Incubating cultures of Escherichia coli with propionic acid (5 mmol/l) or formic acid (10 mmol/l) at pH 5.0 produced bacteriostasis lasting 30 and 120 min respectively. During this time rates of RNA, DNA, protein, lipid and cell wall synthesis were reduced. Growth resumed after continued incubation in the presence of acid, but cells from acid-treated cultures were larger than controls. DNA synthesis was particularly sensitive to the presence of the propionic or formic acid.