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Effect of high-cell-density fermentation of Candida utilis on kinetic parameters and the shift to respiro-fermentative metabolism.

Candida utilis NRRLY-900 was grown in a high-cell-density continuous culture without oxygen limitation. Glucose or molasses was used as carbon source at 30 g l(-1) or 100 g (reducing sugars) l(-1). At 30 g glucose l(-1), the dilution rate (D) immediately before the change in respiratory metabolism (Dr) was approximately 0.40 h(-1). At this value of D, the corresponding culture in molasses did not reach the Dr value. When the reducing sugar concentration in the feed was 100 g l(-1), the Dr was 0.15 h(-1) for glucose and 0.3 h(-1) for molasses. When D>Dr, accumulation of ethanol and organic acids occurred, due to physiological changes in C. utilis. The changes observed were a decrease in the biomass yield coefficient per gram of oxygen consumed (YO2) and a sudden increase in the specific oxygen consumption rate (qO2) for each substrate. Therefore, at growth rates above Dr in a high-cell-density culture, C. utilis acquired a flexible catabolism directed towards alternative fermentation routes. The D at which metabolic changes took place seemed to depend on the nature and concentration of the carbon source. Biomass productivity was higher with molasses than with glucose when the fermenter was operated at high D values.

Acetic Acid↗

Packed bed column fermenter and kinetic modeling for upgrading the nutritional quality of coffee husk in solid-state fermentation.

Studies were carried out to evaluate solid-state fermentation (SSF) for the upgradation of the nutritional quality of coffee husk by degrading the caffeine and tannins present in it. SSF was carried out by Aspergillus niger LPBx in a glass column fermenter using factorial design experiments and surface response methodology to optimize bioprocess parameters such as the substrate pH and moisture content and aeration rate. The first factorial design showed that the moisture content of the substrate and aeration rate were significant factors for the degradation of toxic compounds, which was confirmed by the second factorial design too. The kinetic study showed that the degradation of toxic compounds was related to the development of the mold and its respiration and also to the consumption of the reducing sugars present in coffee husk. From the values obtained experimentally for the oxygen uptake rate and CO(2) evolved, the system determined a biomass yield (Y(x/o)) of 3.811 (g of biomass).(g of consumed O(2))(-1) and a maintenance coefficient (m) of 0.0031 (g of consumed O(2)).(g biomass of biomass)(-1).h(-1). The best results on the degradation of caffeine (90%) and tannins (57%) were achieved when SSF was carried out with a 30 mL.min(-1) aeration rate using coffee husk having a 55% initial moisture content. The inoculation rate did not affect the metabolization of the toxic compounds by the fungal culture. After SSF, the protein content of the husk was increased to 10.6%, which was more than double that of the unfermented husk (5.2%).

Algorithms↗

Cardiolipin is not essential for the growth of Saccharomyces cerevisiae on fermentable or non-fermentable carbon sources.

Cardiolipin is a unique dimeric phospholipid, which is present throughout the eukaryotic kingdom and is specifically localized in mitochondrial membranes. It is widely believed that mitochondria possess an essential requirement for this phospholipid. To determine whether cardiolipin is essential for yeast growth, we generated a cardiolipin synthase null mutant by disrupting the CLS1 gene (open reading frame YDL142c on chromosome IV) of Saccharomyces cerevisiae. Biochemical analysis of the mutant indicated that it had no cardiolipin synthase activity and no cardiolipin in its membranes. The enzyme phosphatidylglycerolphosphate synthase, which catalyses the committed step of the cardiolipin pathway, remained unaffected in the null mutant. Haploid cells containing the null allele are viable in media containing glucose, galactose or glycerol/ethanol as the sole carbon source, although growth in galactose or glycerol/ethanol is somewhat reduced in the mutant compared with the wild type. These results indicate that cardiolipin is not essential for the growth of S. cerevisiae in fermentable or non-fermentable carbon sources.

Amino Acid Sequence↗

[The influence of different straw-based ration type on fermentation and production of volatile fatty acids in the rumen of lactating cows. 3. Rumenal fermentation processes and milk yield criteria after using a low-fiber straw pellet batch as basal ration component and a fully pelleted straw-based ration].

Lactating Black-Pied (DSR) cows were used to study, over a period of 100 days, the influence of different basal ration types (I-treated wheat straw pellets, low-fibre; II- monodiet pellets containing 48% of wheat straw; III = conventional rations based on fresh forage and arable root crops) on the molar fermentation pattern and the daily rate of VFA production and some milk yield criteria. Ration types I and II results in almost equal fermentation patterns of the VFA; as against the conventional ration types (III) a significantly higher molar acetate propertion as well as a highly significantly decreased molar proportion of iso-acids were found. The daily rated of VFA production relativated to energy intake were as follows (in the order of the ration types mentioned): 4.9, 4.2 and 5.0, and 3.3, 2.5 and 3.3 moles per 1,000 anergetic feed equivalents (cattle) after 30 and 100 days of feeding, respectively. In both trials, only about 80% of the relative VFA production of the straw pellet-concentrate (I) and conventional (III) ration types were found when feeding fully pelleted diets (II). On account of the relatively wide variation of the experimental results no statistically secured findings were obtained as to the milk yield criteria, but ration type II produced some 200 and 300 kg milk less thant ration types I and III, respectively, during the 100-day experimental period.

Animal Feed↗

Haloanaerobium fermentans sp. nov., a strictly anaerobic, fermentative halophile isolated from fermented puffer fish ovaries.

A strain of strictly anaerobic and moderately halophilic bacteria isolated from salted puffer fish ovaries was studied phenotypically, genotypically and phylogenetically. On the basis of its physiological and morphological characteristics, the new isolate is considered to be a member of the genus Haloanaerobium. It is a motile, rod-shaped, non-spore-forming, gram-negative, obligate anaerobe that grows in the presence of 25% (w/v) NaCl. The optimum salt concentration for growth is 10% (w/v). It grows well at 15 and 45 degrees C, but not at 10 or 50 degrees C. The optimum temperature for growth is 35 degrees C. It grows at pH 6.0-9.0 and the optimum pH for growth is 7.5. It ferments N-acetylglucosamine, cellobiose, fructose, galactose, D-glucose, lactose, maltose, D-mannose, raffinose, D-ribose, sucrose and D-xylose. It ferments D-glucose with the production of hydrogen, carbon dioxide, ethanol and organic acids such as acetate, formate and lactate. 16S rRNA gene sequence information confirmed the phylogenetic position of the new isolate, strain R-9T, as a member of the genus Haloanaerobium. DNA-DNA hybridization data revealed that isolate R-9T exhibited low levels of reassociation (less than 30%) with previously described Haloanaerobium species. Based on these results, the new isolate appears to represent a new Haloanaerobium species, for which the name Haloanaerobium fermentans sp. nov. is proposed. The type strain is R-9T (= JCM 10494T).

Acetylglucosamine↗

Lactobacillus frumenti sp. nov., a new lactic acid bacterium isolated from rye-bran fermentations with a long fermentation period.

Within the framework of the characterization of the microflora of an industrial sourdough fermentation, strains of Lactobacillus amylovorus, Lactobacillus pontis and two other strains were isolated which could not be associated with a valid species. These latter strains were Gram-positive, catalase-negative, non-spore-forming, non-motile rods that could be clearly differentiated from known species by 16S rDNA sequence analysis. For further characterization, the morphological, physiological (sugar fermentation, formation of DL-lactate, hydrolysis of arginine, growth temperature, CO2 production) and chemotaxonomic (G+C content, cell wall composition, SDS-PAGE of whole-cell proteins) properties were determined. Fitting of the complete 16S rDNA sequence into alignments of such sequences, together with the subsequent phylogenetic calculations, allowed the reconstruction of a phylogenetic tree. These data showed that the two strains were phylogenetically related but formed an independent cluster distinct from their closest neighbours, L. pontis, Lactobacillus panis, Lactobacillus oris, Lactobacillus vaginalis and Lactobacillus reuteri. The results of DNA-DNA hybridization experiments indicated that the two isolates represent a new Lactobacillus species, for which the name Lactobacillus frumenti is proposed; the type strain of this species is DSM 13145T (= LMG 19473T).

Bread↗

Pyruvate fermentation by Oenococcus oeni and Leuconostoc mesenteroides and role of pyruvate dehydrogenase in anaerobic fermentation.

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate --> 1 lactate + 1 acetate + 1 CO(2)). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.

Anaerobiosis↗

Microbial fermentation of rice straw: nutritive composition and in vitro digestibility of the fermentation products.

Rice straw was fermented with Cellulomonas sp. and Alcaligenes faecalis. Microbial cells and undigested residue, as well as chemically treated (NaOH or NH4OH) and untreated straws, were analyzed for nutrient composition and in vitro digestibility. In a typical fermentation, 75% of the rice straw substrate was digested, and 18.6% of the total substrate weight that disappeared was recovered as microbial protein. The microbial cell fraction was 37% protein and 5% crude fiber; the residue was 12% protein and 45% crude fiber. The microbial protein amino acid profile was similar to alfalfa, except for less cysteins. The microbial cells had more thiamine and less niacin than Torula yeast. In vitro digestibility of the microbial protein was 41.2 to 55%, that of cellulose was 52%.

Actinomycetales↗

Pyrazinamidase, CR-MOX agar, salicin fermentation-esculin hydrolysis, and D-xylose fermentation for identifying pathogenic serotypes of Yersinia enterocolitica.

We evaluated several simple laboratory tests that have been used to identify pathogenic serotypes of Yersinia enterocolitica or to indicate the pathogenic potential of individual strains. A total of 100 strains of Y. enterocolitica were studied, including 25 isolated during five outbreak investigations, 63 from sporadic cases, and 12 from stock cultures. The pyrazinamidase test, which does not depend on the Yersinia virulence plasmid, correctly identified 60 of 63 (95% sensitivity) strains of pathogenic serotypes and 34 of 37 (92% specificity) strains of nonpathogenic serotypes. Salicin fermentation-esculin hydrolysis (25 degrees C, 48 h) correctly identified all 63 (100% sensitivity) strains of the pathogenic serotypes and 34 of 37 (92% specificity) strains of the nonpathogenic serotypes. The results of the pyrazinamidase and salicin-esculin tests disagreed for only 7 of the 100 strains of Y. enterocolitica, and these would require additional testing. Congo red-magnesium oxalate (CR-MOX) agar determines Congo red dye uptake and calcium-dependent growth at 36 degrees C, and small red colonies are present only if the strain contains the Yersinia virulence plasmid. This test has proven to be extremely useful for freshly isolated cultures, but only 15 of 62 strains of pathogenic serotypes that had been stored for 1 to 10 years were CR-MOX positive. None of the 16 strains of Y. enterocolitica serotype O3 fermented D-xylose, so this test easily differentiated strains of this serotype, which now appears to be the most common in the United States. Although antisera that can actually be used to serotype strains of Y. enterocolitica are not readily available, the four simple tests described above can be used to screen for pathogenic serotypes.

Agar↗

Evaluation of Vitek GNI+ and Becton Dickinson Microbiology Systems Crystal E/NF identification systems for identification of members of the family Enterobacteriaceae and other gram-negative, glucose-fermenting and non-glucose-fermenting bacilli.

We evaluated the Vitek GNI+ and Becton Dickinson Crystal E/NF identification systems for their ability to accurately identify 619 and 626 strains, respectively, of members of the family Enterobacteriaceae and other glucose-fermenting and non-glucose-fermenting gram-negative rods. All strains tested were taken from a stock collection and passed three times on 5% sheep blood agar prior to testing. These strains represented a more rigorous challenge to both systems than one resulting from the testing of consecutive clinical isolates. Testing with both systems was done according to the manufacturers' instructions, and tests were repeated in duplicate when errors occurred. Vitek version 5.01 and Crystal version 3.0 softwares were used for identifications. The identification results from each system were compared with identifications previously determined with reference biochemicals. At the completion of the appropriate incubation period, the GNI+ and Crystal systems correctly identified 80.1 and 71.1% of the total isolates, respectively. After additional tests suggested by the software programs were completed, the GNI+ had an accuracy of 87.6% and the Crystal system's accuracy had improved to 87.9%. The error rates for the GNI+ and Crystal systems were 6.5 and 5.3%, respectively. A report of "no identification" was given for 6.0 and 6.9% of the isolates, respectively, and was associated with no particular organism group. One isolate each of Acinetobacter lwoffii and Vibrio alginolyticus would not grow in the Vitek card. The average times to detection for correct enteric identifications in the GNI+ system were 4.1 and 6.8 h for nonenteric identifications, while the Crystal results were routinely read at 18 h. We conclude that there was no significant difference (P > 0.05) between the results of the GNI+ card and those of the Crystal E/NF system after additional testing was performed with the group of organisms tested, but the overall accuracy for both systems in this study was below 90%.

Bacterial Typing Techniques↗

Nissui glucose fermentative gram-negative rod identification system EB-20 gives a unique profile for typical non-sorbitol-fermenting Escherichia coli O157:H7.

The 98 non-sorbitol-fermenting (NSF) Escherichia coli O157:H7 strains identified on a Nissui glucose fermentative gram-negative rod identification system (EB-20) gave a unique biochemical profile number that was not detected in 85 pathogenic and 13 nonpathogenic E. coli strains. Thus, EB-20 is useful for the identification of NSF E. coli O157:H7 and provides a simple, cost-effective, and reliable tool for clinical laboratories.

Escherichia coli O157↗

Identification of Gram-negative non-fermenters and oxidase-positive fermenters by the Oxi/Ferm tube.

Since the recent introduction of the Roche Oxi/Ferm Tube to the UK two identification schemes have been developed by the manufacturer for use with the kit. We evaluated the success of these two schemes in identifying 222 predominantly culture collection strains belonging to 45 taxa of non-fermenters and nine taxa of oxidase-positive fermenters. The strains were chosen to represent all the taxa included in the two identification schemes developed by the manufacturer and we have therefore been able to assess the overall success of identification by the two schemes. Since, however, our choice of strains does not reflect their incidence in clinical material, our identification rates are not necessarily those that might be obtained in a routine clinical laboratory. The most advanced identification scheme so far developed for the Oxi/Ferm Tube (CCIS System 1977-1432) allowed 62% of the 222 strains to be correctly identified although a disturbing feature was that more of the strains that were not correctly identified were incorrectly identified (24%) rather than not identified (14%); these figures represent an improvement over the earlier identification scheme (CCIS System 1976-621-74346) for which the corresponding figures were 56%, 32%, and 12%. CCIS System 1977-1432 seems likely to give a better performance in a routine clinical laboratory than in this study since for those taxa which, we would judge from the material sent to us for identification, are most commonly seen in a routine laboratory (Acinetobacter calcoaceticus, A. lwoffii, Pseudomonas aeruginosa, P. fluorescens, P. maltophilia, P. pseudoalcaligenes, and P. putida) 89% were correctly identified, none remained unidentified, and 11% were incorrectly identified. Thirty strains, each of a different taxon, were tested in triplicate to assess the reproducibility of reactions in the Oxi/Ferm Tube.

Bacteriological Techniques↗

Coupling of fermentation and esterification: microbial esterification of decanoic acid with ethanol produced via fermentation.

Two different kinds of bioprocess, ethanol fermentation and subsequent microbial esterification, were coupled using Issatchenkia terricola IFO 0933 in an interface bioreactor. The strain produced ethyl decanoate (Et-DA) by esterification of exogenous decanoic acid (DA) with ethanol produced via fermentation. The efficiency of the new coupling system depended on the concentration of glucose in a carrier and DA in an organic phase (decane) in an agar plate interface bioreactor. Optimum glucose content and DA concentration were 4% and 29 mM, respectively.

Decanoic Acids↗

Effect of magnesium and potassium on microbial fermentation in a continuous culture fermentation system with different levels of monensin or lasalocid.

Two continuous culture fermentation experiments were used to determine ruminal microbial fermentation in the presence of monensin (Exp. 1) or lasalocid (Exp. 2) with different dietary concentrations of Mg and K. In each experiment, eight 500-ml continuous culture fermentors were supplied with late-cut coastal bermuda grass (80%) and cellulose (20%) as substrate during five trials with two levels of Mg (.07 and .14%), K (.6 and 2.5%) and either monensin (Exp. 1; 0 and 20 mg/kg) or lasalocid (Exp. 2; 0 and 20 mg/kg) in a 2 x 2 x 2 factorial arrangement of treatments (each trial served as a replication). Substrate was added every 8 h (5 g/fermentor) for 5 d and digesta samples were collected every 2 h for 6 h and at 20 h for volatile fatty acid (VFA) analysis on d 6. In Exp. 1, monensin decreased (P less than .05) the acetate:propionate ratio (A:P) from 2.98 to 2.22. A Mg X monensin interaction was observed for the molar percentage of acetate and propionate (P less than .05). There was a 4.8% reduction in acetate (mol/100 mol) when monensin was fed with .07% Mg and a 12.6% decrease (P less than .01) when fed with .14% Mg compared with diets not containing monensin. Propionate (mol/100 mol) increased 14% when monensin was fed with .07% Mg and 32% (P less than .001) when fed with .14% Mg compared with diets not containing monensin. Potassium did not affect VFA concentrations in the presence of monensin or Mg. In Exp. 2, lasalocid decreased (P less than .01) the A:P ratio from 3.53 to 2.86.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗