Effect of the degree of dilution of the antibiotic fermentation broths on the filtration indices]

Dilution of the fermentation broths with water before the mycelium separation lowered the specific cake resistance. The effect of the dilution on the filtration duration was different and depended on the fermentation broth type. As for the erythromycin fermentation broth, the time of its filtration decreased after the dilution, while the filtration time of the fermentation broths of the other 2 antibiotic-producing organisms increased after the dilution.     

Citric acid production

Selective separation of citric acid and process optimization for its recovery from fermented broth through precipitation and acid hydrolysis were tried. A mathematical model has been proposed for yield. The experimental values are in good agreement with predicted values; the correlation coefficient was found to 0.9198.     

Ultrafiltration of fermented broths and solvent extraction of antibiotics

Experiments of ultrafiltration with diafiltration were performed on fermented broths of benzylpenicillin and clavulanic acid produced at pilot scale at CIPAN, SA to achieve solid/liquid separation, antibiotic recovery and soluble protein retention. This study was done using a laboratorial/pilot scale ultrafiltration facility from Paterson Candy International Ltd. whose modules have a tubular configuration. PVDF membranes with a molecular weight cut-off of 100 000 (FP10) and 200 000 (FP200) were assessed. Comparison between solvent extraction of benzylpenicillin and clavulanic acid from filtered broth and ultrafiltered broth was also done. It was concluded that ultrafiltration with diafiltration of fermented broths of antibiotics using the FP10 membrane is a valid alternative to the use of rotary vacuum filters, flocculants and filter aid, deserving a further study to establish the best operating conditions. An economic appraisal also seems to be of paramount importance.To CIPAN-SA in which this work was done and where the fermented broths were produced. To JNICT/FSE for the funding of the project in the scope of which this work was done.     

Morphological measurements on Penicillium chrysogenum broths by rheology and filtration methods

The morphology parameters of mycelial culture (Penicillium chrysogenum) were measured and quantified by rheology and filtration methods. Two of the morphology parameters obtained from rheology measurements, delta defined by the Casson equation and delta* defined by intrinsic viscosity, were found to vary systematically with broth age and with the observed morphology by microscopy. Three of the filtration parameters, hyphal density, Kozeny constant, and index of compressibility, are demonstrated as sensitive indicators of the broth age and mycelial morphology. Two of the morphology parameters, delta and delta*, were used to cross-correlate with hyphal density. Because various mycelial fermentations require different growth morphologies (pellet and filament) for optimum product yield and the morphology of mycelial broths varies with broth age, it is suggested that these morphology parameters could be used to represent the morphology of mycelial broths quantitatively. (c) 1993 John Wiley & Sons, Inc.     

Development of aqueous two‐phase systems comprising poly ethylene glycol and dextran for purification of pullulan: Phase diagrams and fiscal analysis

Pullulan is a commercially important Exopolysaccharide (EPS) with wide‐spread applications which is produced by Aureobasidium pullulans. The alternative α (1 4) & α (1 6) configuration in pullulan provides it the specific structural and conformational properties. Pullulan is currently being exploited in food, health care, pharmacy, lithography, cosmetics. The fermented broth is processed by organic solvent precipitation for isolation and purification of pullulan. In this study, we have tried to analyze the potential of aqueous two phase system as an alternate technique to extract pullulan from fermented broth. Including this viability of ATPS was also compared with conventional organic solvent precipitation system in terms of cost and time. It was found that ATPS process produced a higher yield of pullulan (80.56%) than organic solvent precipitation method (71.6%). ATPS was also found more economical and less time consuming method.     

Development of Suitable Solvent System for Downstream Processing of Biopolymer Pullulan Using Response Surface Methodology

Downstream processing is an important aspect of all biotechnological processes and has significant implications on quality and yield of the final product. Several solvents were examined for their efficacy on pullulan precipitation from fermentation broth. Interactions among four selected solvents and their effect on pullulan yield were studied using response surface methodology. A polynomial model was developed using D-optimal design and three contour plots were generated by performing 20 different experiments and the model was validated by performing optimization experiments. The results indicated that lower concentration of ethanol in combination with the other three solvents has resulted in higher yield of polymer from fermentation broth and the optimized solvent system was able to recover 1.44 times more pullulan as compared to the conventional ethanolic precipitation method. These observations may help in enhancing efficiency of pullulan recovery from fermentation broth and also result in reduced cost of production for the final product.     

Direct inoculation into media containing bile salts and antibiotics is unsuitable for the detection of acid/salt stressed Escherichia coli O157:H7

The efficiency of selective enrichment broths for the recovery of low numbers of acid/salt stressed Escherichia coli O157:H7 was determined. Stressed cultures were diluted to low levels and recovered in tryptone soya broth with added bile salts, to make modified tryptone soya broth, and buffered peptone water with various combinations of antibiotic supplementation including novobiocin, acriflavine and a mixture of vancomycin, cefsulodin and cefixime (VCC) at 37 °C and 42 °C. Significantly fewer stressed cells, in some cases as little as 0·3% of the starting population, were recovered by all the selective enrichment broths containing bile salts or VCC antibiotics compared to the non-selective controls. The use of such enrichments to recover low numbers of stressed E. coli O157:H7 may result in failure to detect the organism. Parallels with salmonella methodology are made and the need for a non-selective pre-enrichment stage in E. coli O157:H7 methods discussed.     

Comparison of some enrichment broths and growth media for the isolation of thermophilic campylobacters from surface water samples

Three different enrichment broths and two selective growth media were compared for isolating thermophilic campylobacters by combined membrane filtration and enrichment techniques from surface waters of different physical, chemical and bacteriological characteristics. Fifty-two strains of campylobacters were isolated from total of 1668 cultures. The various broth/medium combinations did not affect the dominance of C. jejuni over C. coli (total 49 C. jejuni and three C. coli). The most efficient combinations of enrichment broth and growth media were either Oosterom broth/blood-free charcoal-cefoperazone-deoxycholate agar (CCDA) medium or blood-free charcoal-cefoperazone-deoxycholate (CCD) broth/CCDA medium. Modified Preston broth (sheep blood instead of horse blood) with either of the growth media gave significantly lower yields although it suppressed efficiently the growth of contaminants. Skirrow medium had lower selectivity than CCDA medium and gave slightly lower isolation rate. Enrichment time (24 or 48 h) did not affect the isolation frequency of campylobacters but longer enrichment time increased the growth of contaminants. Prefiltration through membranes of pore sizes 5.0 and 1.2 microns decreased the growth of contaminants. However, these membranes retain campylobacters and must be cultured to avoid underestimation. From more polluted waters campylobacters were isolated most frequently with CCD broth and CCDA medium.     

Pre-Enrichment and Enrichment Methods for Isolating Small Number of Campylobacteria from Contaminating Flora

A method was developed to detect fewer than 100 CFU of campylobacteria from SIFF transport medium to which thawing drip from deep frozen broiler carcasses was added as a source of contamination and which was then stored at room temperature for 20 h. The method was made possible by using pre–enrichment in 1 % buffered peptone water under a microaerophilic atmosphere for 5 h at 43°C before selective enrichment either in brucella enrichment broth and on brucella blood selective agar supplemented with Skirrow antibiotics or in CCD enrichment broth and on blood free CCD selective agar. The other pre–enrichment broth studied was alkaline peptone water with reducing agents (RAPW) and the other enrichment broths and selective agars were Preston broth and agar, THAL broth and alkaline tryptose broth (ATB) and brucella agar with ATB antibiotics. Contaminating flora can be a problem when using enrichment broths and selective agars with limited antibiotic supplementation.     

To the problem of Campylobacter jejuni detectability in water

In a series of model experiments two isolation procedures for the detection of water-borne Campylobacter jejuni were compared: a standard culture in thioglycolate broth enriched with 7% defibrinated sheep blood and supplement C and a modified membrane filtration method in which the filter (porosity 0.45 microns) plated on campylobacter agar surface was removed after the first 24 hours of incubation and the plate further incubated for 48 hours. The recovery rates by the thioglycolate broth method were markedly less pronounced than those obtained by the modification of membrane filtration technique, especially in the case of water rich in organics. The best isolation parameters were achieved with water samples of at least 10 ml in volume.     

Optimising recovery of Campylobacter spp. from the lower porcine gastrointestinal tract

To determine the incidence of campylobacters in Northern Ireland pigs, ileal contents and anal swabs were taken shortly after death. Direct streaking onto Preston agar, and modified charcoal cefoperazone desoxycholate agar (mCCDA), were compared, as was enrichment in selective broths prior to streaking onto the corresponding solid medium. For anal swabs direct plating on mCCDA was most efficient, with 100% of samples positive, whilst for ileal contents enrichment in mCCD broth was best with 86% of samples positive. Although only 34% of ileal samples enriched in Preston broths were positive they yielded three species not isolated from mCCD broth, and hence indicated that some pigs were infected by at least two species of Campylobacter. Overall, the number of samples found to contain campylobacters, and the range of species isolated, was seen to be markedly affected by both the choice of selective medium and the isolation procedures.     

Preparative high-performance liquid chromatographic separation and analysis of the Maltacine complex - a family of cyclic peptide antibiotics from Bacillus subtilis

Purification of secondary metabolites from fermentation broths can be a challenging task both due to the complexity of the medium, inherently unstable molecular structures or by the action of enzymes present in the fermentation broth leading to poor isolation yield and loss of antibiotic activity. A combination of different purification techniques has usually been used to arrive at acceptable purities for characterisation of the target molecules. Due to rapid decay of antimicrobial activity a rapid preparative high-performance liquid chromatography (HPLC) method was developed that provided separation and resolution of a family of 18 closely related cyclic peptides within 110 min with minimal loss of activity. Characterisation of the peptides with LC-MS, UV/IR spectroscopy and amino acid analysis disclosed 20 different peptides with cyclic structures (lactones) with molecular weights between 1447.7 and 1519.8 Da. No peptide antibiotics with identical molecular weights have previously been reported in the literature, which lead us to conclude that this peptide complex has not been discovered before. We have named them Maltacines.     

Determination of the filtration characteristics of the fermentation broths of antibiotic producers]

Main filtration characteristics of the fermentation broths are necessary for estimation of the filtration equipment, rational choice of the filtering apparatus, determination of the optimal conditions for their exploitation. In this connection studies were carried out with a purpose of determining the specific resistance and the content of the solid phase in the fermentation broths of the main antibiotics. It was shown that the above characteristics were closely connected with the biosynthetic conditions and medium composition. It was noted that the specific resistance of the precipitates of the fermentation broths of various antibiotic-producing organisms significantly differed, sometimes by several orders. Preliminary treatment of the fermentation broths before filtration provided a marked decrease in the resistance of the precipitate.     

Isolation of Salmonellae from Foods Samples: IV. Comparison of Methods of Enrichment

A comparison of various methods of enhancing frequency of Salmonella isolations revealed that inoculation of a second enrichment broth, with culture from the first, was no improvement over the single direct enrichment method. It was inferior to centrifugation.

Selenite was observed to produce more positive isolations at 48 hr than at 24. No change occurred in tetrathionate. Reconstitution of dried albumen with water produced a significant increase in isolations over direct inoculation of enrichment broth in the case of tetrathionate but not selenite broth.

Pre-enrichment in lactose broth before inoculation of enrichment media was vastly superior to reconstitution in water for both enrichment broths. A comparison of results obtained using dulcitol, mannitol, lactose and carbohydrate-free purple broths in pre-enrichment indicated that the carbohydrate added was immaterial.

     

Rapid method for monitoring maximum biosurfactant production obtained by acetone precipitation

Summary The appearance of surface active compounds in the culture broths grown on hydrocarbons can be easily monitored by measuring the surface tension of the cell free broth. Using three bacterial strains it was found that yields of the biosurfactants were maximum at the time when lowest surface tension values were recorded. Thus reduction of surface tension of the medium is a rapid method for assay of maximum biosurfactant formation prior to their actual isolation.     

Increased recovery of salmonellae from environmental samples enriched with buffered peptone water.

The incidence and persistence of salmonellae in weather pools on the top of Stone Mountain were investigated with lactose and buffered peptone water used as pre-enrichment broths. A total of 162 samples were collected from 16 weather pools over a 3-month period. The use of buffered peptone water increased the recovery of salmonellae by approximately 25%. The combined use of direct enrichment in tetrathionate broth containing brilliant green dye and pre-enrichment in buffered peptone water followed by enrichment in tetrathionate broth made it possible to detect all 37 of the contaminated samples. All of the isolates were Salmonella bareilly, the only serotype recovered in a previous study. All but one of the isolations were made from moist or wet samples. S. bareilly was isolated from rabbit dung and litter collected near the weather pools, but attempts to trap rabbits for study were unsuccessful. Random samples taken along a side of the mountain yielded S. bareilly in weather pools within the upper third of the mountain; below this level, S. weslaco and S. memphis were recovered, but not S. bareilly.     

Increased recovery of salmonellae from environmental samples enriched with buffered peptone water.

The incidence and persistence of salmonellae in weather pools on the top of Stone Mountain were investigated with lactose and buffered peptone water used as pre-enrichment broths. A total of 162 samples were collected from 16 weather pools over a 3-month period. The use of buffered peptone water increased the recovery of salmonellae by approximately 25%. The combined use of direct enrichment in tetrathionate broth containing brilliant green dye and pre-enrichment in buffered peptone water followed by enrichment in tetrathionate broth made it possible to detect all 37 of the contaminated samples. All of the isolates were Salmonella bareilly, the only serotype recovered in a previous study. All but one of the isolations were made from moist or wet samples. S. bareilly was isolated from rabbit dung and litter collected near the weather pools, but attempts to trap rabbits for study were unsuccessful. Random samples taken along a side of the mountain yielded S. bareilly in weather pools within the upper third of the mountain; below this level, S. weslaco and S. memphis were recovered, but not S. bareilly.     

Eight enrichment broths for the isolation of Campylobacter jejuni from inoculated suspensions and ground pork

Aims:  The efficiency of eight enrichment broths for the selective isolation of Campylobacter jejuni was compared to identify an optimal enrichment broth.
Methods and Results:  Brucella-FBP, Preston, Doyle and Roman, modified CCD (mCCD), Park and Sanders, Bolton, Hunt and Radle and Hunt broths were compared for their recovery of (i) Camp. jejuni in suspension, (ii) Camp. jejuni from inoculated ground pork, (iii) heat-injured Camp. jejuni (55°C for 20 min) in suspension and (iv) heat-injured Camp. jejuni from inoculated ground pork. Hunt broth and Bolton broth showed the highest and most rapid enrichment efficacy for the cell suspensions and ground pork, respectively. Preston, Park and Sanders and mCCD broths had relatively high enrichment efficiencies, while Brucella-FBP broth was significantly inferior to the other broths ( P  < 0·05).
Conclusions:  Cell recovery from the eight enrichment broths was dependent on the sample type and the state of the cells. The use of the appropriate broth is important for the rapid and efficacious enrichment of Camp. jejuni . In particular, heat-injured Camp. jejuni require a longer cultivation time and a suitable enrichment broth.
Significance and Impact of the Study:  The results from the present study provide information for selecting the most appropriate enrichment broth for Camp. jejuni and may contribute to improved detection methods for the organism.     

Optimizing enrichment conditions for the isolation of Escherichia coli O157 in soils by immunomagnetic separation

AIMS: To compare a range of enrichment broths and enrichment temperatures for the isolation of Escherichia coli O157 by immunomagnetic separation (IMS) from sandy, loam and clay soils. METHODS AND RESULTS: Soils were spiked with cocktails of four atoxigenic strains of E. coli O157 and four strains of commensal E. coli. The organisms were stressed by subjecting soils to cycles of freeze/thawing, followed by drying at 20 degrees C for up to 4 days. Nine enrichment broths were trialled based on buffered peptone water, tryptone soya broths and EC broths supplemented with a range of selective additions. Enrichments were incubated for 6 h and assessed by target recovery after IMS on cefixime tellurite sorbitol MacConkey agar (CTSMAC) incubated at 37 degrees C for 24 h. A comparison of enrichment temperatures (37 and 42 degrees C) was also performed. Buffered peptone water (with or without vancomycin) and tryptone soya broth (with or without novobiocin) gave significant increases in recovery of E. coli O157 compared to others tested. In addition, broths incubated at 42 degrees C were superior to those at 37 degrees C for the recovery of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that sub-lethally damaged E. coli O157 surviving in soil can be sensitive to antimicrobial additions. The choice and concentration of these additions is vitally important to optimize target recovery. Some IMS protocols, established for the isolation of E. coli O157, may be unsuitable for the examination of soil samples.     

豚草种带内生真菌及其对种子发芽和幼苗生长的影响

内生真菌是一类共生于植物体内,能够不同程度影响宿主植物生态适应性和竞争能力的微生物。分析内生真菌在豚草种子中的分布、种群结构,以及内生真菌发酵液对种子发芽和幼苗生长的作用。结果显示:发生于6个地区的豚草种子均能分离获得内生真菌,分离率在19%—92.63%之间,不同地区之间差异极显著(P0.01)。内生真菌主要存在于种子的总苞部位,分离率达到65.52%。发生于福建省长乐市松下镇的豚草种带内生真菌种群包含5个属,以链格孢属(Alternaria)真菌为优势菌群,占82.26%;其次为镰孢属(Fusarium)真菌,占9.68%;其它3个属的真菌发生较少,均低于5%。内生真菌主要以水平传播方式在豚草不同世代之间传播。供试的7个内生真菌菌株的发酵液均不同程度地抑制豚草种子发芽,降低幼苗地上部高度、根长度、根数量和总生物量,但不同菌株发酵液之间抑制程度差异明显,显示不同菌株对豚草种子发芽和幼苗生长产生不同的影响。内生真菌发酵液处理后的种子仍然保持较高程度的活力;不同内生真菌发酵液处理后,有活力的种子维持在50%—87.5%之间,均高于(或等于)清水处理的种子,说明内生真菌代谢产物只是抑制种子的发芽,但并不导致种子的腐烂和死亡。这些研究结果初步显示种子携带的内生真菌可能在豚草入侵生物学中发挥重要的作用。