Production and properties of an alkaline,thermophilic lipase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> NS2W

Eighteen bacterial strains were isolated from soil samples and screened for alkaline, thermophilic lipase production. Pseudomonas fluorescens NS2W was selected and its production of lipase was optimized in shake flasks using a statistical experimental design. Cell growth and lipase production were studied in shake flasks and in a 1-l fermenter in the optimized medium. Maximum lipase yields were 69.7 and 68.7 U ml⁻¹, respectively. The optimized medium resulted in about a five-fold increase in the enzyme production, compared to that obtained in the basal medium. The lipase had an optimal activity at pH 9.0 and was stable over a wide pH range of 3–11 with more than 70% activity retention. The lipase had an optimal activity at 55°C and was stable up to 60°C with more than 70% activity retention for at least 2 h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 344–348 DOI: 10.1038/sj/jim/7000254 Received 06 September 2001/ Accepted in revised form 15 March 2002     

Production of H2 by sulphur-deprived cells of the unicellular cyanobacteria Gloeocapsa alpicola and Synechocystis sp. PCC 6803 during dark incubation with methane or at various extracellular pH

AIMS: To examine sulphur (S) deprivation in combination with the presence of methane (CH4) and changes in extracellular pH as a method to enhance in situ hydrogen (H2) generation during fermentation in the unicellular non-diazotrophic cyanobacteria Gloeocapsa alpicola and Synechocystis PCC 6803. METHODS AND RESULTS: The level of H2 production, measured using a gas chromatography, was determined in S-deprived cells of G. alpicola and Synechocystis PCC 6803 during fermentation. Starvation on S enhanced the rate of H2 production by more than fourfold in both strains. S-deprived cyanobacteria were able to maintain maximum rate of H2 production during at least 8 h of fermentation representing the entire dark period of a day. Increased H2 production was observed during dark anoxic incubation with a gas phase of 100% CH4 (up to four times) at lower pH of the medium (5.0-5.5). CONCLUSIONS: S-deprivation in combination with CH4, added or maybe produced by another micro-organisms, and changes in the pH of the media can be used to further increase the specific capacity of unicellular non-N2-fixing cyanobacteria to produce H2 during fermentation with the overall aim of applying it for outdoor photobiological H2 production. SIGNIFICANCE AND IMPACT OF THE STUDY: S-deprivation with respect to H2 production is well studied in the green algae Chlamydomonas reinhardtii while its application for H2 production in cyanobacteria is novel. Similarly, the stimulation of H2 generation in the presence of CH4 opens up new possibilities to increase the H2 production. Natural gas enriched with H2 seems to be a perspective fuel and may be an intermediate step on the pathway to the exploitation of pure biohydrogen.     

Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillus flavus

AIMS: To find a supplemental ingredient that can be added to routinely used growth media to increase conidial production and decrease aflatoxin biosynthesis in small sclerotial (S strain) isolates of Aspergillus flavus. METHODS AND RESULTS: Molasses was added to three commonly used culture media: coconut agar (CAM), potato dextrose agar (PDA), and vegetable juice agar (V8) and production of conidia, sclerotia, and aflatoxins by A. flavus isolate CA43 was determined. The effect of nitrogen sources in molasses medium (MM) on production of conidia, sclerotia and aflatoxins was examined. Water activity and medium pH were also measured. Conidia harvested from agar plates were counted using a haemocytometer. Sclerotia were weighed after drying at 45 degrees C for 5 days. Aflatoxins B(1) and B(2) were quantified by high-performance liquid chromatography. Addition of molasses to the media did not change water activity or the pH significantly. Supplementing CAM and PDA with molasses increased conidial production and decreased aflatoxins. Two-fold increased yield of conidia was found on MM, which, like V8, did not support aflatoxin production. Adding ammonium to MM significantly increased the production of sclerotia and aflatoxins, but slightly decreased conidial production. Adding urea to MM significantly increased the production of conidia, sclerotia and aflatoxins. CONCLUSIONS: Molasses stimulated conidial production and inhibited aflatoxin production. Its effect on sclerotial production was medium-dependent. Water activity and medium pH were not related to changes in conidial, sclerotial or aflatoxin production. Medium containing molasses alone or molasses plus V8 juice were ideal for conidial production by S strain A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into molecular events associated with the utilization of molasses may help to elucidate the mechanism(s) that decreases aflatoxin biosynthesis. Targeting genetic parameters in S strain A. flavus isolates may reduce aflatoxin contamination of crops by reducing the survival and toxigenicity of these strains.     

Description of conidia from submerged cultivation of Thermomyces lanuginosus for use as a uniform inoculum

Outer membranes were isolated, by sodium lauryl sulphate extraction, from the American type strain, five Australian, and four English isolates of Campylobacter hyointestinalis. On SDS-PAGE examination, the protein profiles of seven strains (including the type strain) were similar, and were dominated by two major proteins of 47 and 50 kDa. Three other isolates had unique major protein profiles. The largest of these proteins was heat-modifiable in these isolates, and in the type strain. The flagellin of three isolates screened was of similar M(r) to that of Campylobacter jejuni/Campylobacter coli. The lipopolysaccharides of C. hyointestinalis isolates were heterogeneous in structure; 5/10 isolates synthesised material of M(r) value greater than that of the low M(r) C. jejuni/C. coli lipopolysaccharide. By gel excision and re-electrophoresis, it was shown that the higher M(r) materials of one isolate were not artifactual aggregates of lower M(r) species.     

Isolation and characterization of sulphur-oxidizing Thiomonas sp. and its potential application in biological deodorization

AIMS: To isolate and characterize a sulphur-oxidizing bacterial strain from activated sludge and to evaluate its potential application in biological deodorization. METHODS AND RESULTS: A dominant sulphur-oxidizing bacterial strain, designated as strain SS, was isolated from an enrichment culture using thiosulphate as a sole energy source and CO2 as a sole carbon source. The cells of this organism were aerobic, rod-shaped, Gram-negative and motile. Strain SS could grow autotrophically, heterotrophically as well as mixotrophically. Autotrophic growth was observed at pH values ranging from 2.3 to 9.0. Phylogenetic analyses revealed that strain SS belonged to Group 1 of the genus Thiomonas, closely related to Thiomonas perometabolis and Thiomonas intermedia. The thiosulphate oxidation rates of strain SS at different pH values were evaluated in terms of oxygen uptake using a Micro-Oxymax respirometer. The results showed that the maximum oxidation rate of 5.65 mg l(-1) h(-1) occurred at 56 h of growth and pH 6.0. Continuous H2S removal study demonstrated that strain SS could remove more than 99% of H2S when the inlet concentration was below 58.6 ppm. Further increase of the inlet concentration to 118 ppm gave rise to a decline in the removal efficiency to ca 90%. CONCLUSIONS: The strong acidification of the culture medium during the later period could result in the deterioration of the growth activity and the metabolism activity of strain SS. In practical application, the problems caused by the end-product inhibition and the acidification can be alleviated by periodical replacement of culture medium with fresh medium. Given the physiological flexibility and the ability to remove H2S rapidly and efficiently, strain SS could be a good 'deodorizing' candidate. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that Thiomonas species has been reported for biological deodorization application.     

Enhancement of acid amylase production by an isolated Aspergillus awamori

AIMS: Evaluation of the influence of fermentation components on extracellular acid amylase production by an isolated fungal strain Aspergillus awamori. METHODS AND RESULTS: Eight fungal metabolic influential factors, viz. soluble starch, corn steep liquor (CSL), casein, potassium dihydrogen phosphate (KH(2)PO(4)) and magnesium sulfate (MgSO(4) x 7H(2)O), pH, temperature and inoculum level were selected to optimize amylase production by A. awamori using fractional factorial design of Taguchi methodology. Significant improvement in acid amylase enzyme production (48%) was achieved. The optimized medium composition consisted of soluble starch--3%; CSL--0.5%; KH(2)PO(4)--0.125%; MgSO(4) x 7H(2)O--0.125%; casein--1.5% at pH 4.0 and temperature at 31 degrees C. CONCLUSION: Optimization of the components of the fermentation medium was carried out using fractional factorial design of Taguchi's L-18 orthogonal array. Based on the influence of interaction components of fermentation, these could be classified as the least significant and the most significant at individual and interaction levels. Least significant factors of individual level have higher interaction severity index and vice versa at enzyme production in this fungal strain. The pH of the medium and substrate (soluble starch) showed maximum production impact (60%) at optimized environment. Temperature and CSL were the least influential factors for acid amylase production. SIGNIFICANCE AND IMPACT OF THE STUDY: Acid amylase production by isolated A. awamori is influenced by the interaction of fermentation factors with fungal metabolism at individual and interaction levels. The pH of the fermentation medium and substrate concentration regulates maximum enzyme production process in this fungal strain.     

Evaluation of a quantitative screening method for hydrogen sulfide production by cheese-ripening microorganisms: the first step towards l-cysteine catabolism

A practical adaptation of the methylene blue reaction for hydrogen sulfide quantification was developed to perform microbial selection. Closed plate flasks containing a zinc-agar layer above the liquid microbial culture are proposed as a trap system where the H(2)S can be retained and then quantified by the methylene blue reaction. Using this quantitative method, the ability to produce H(2)S was studied in several cheese-ripening microorganisms. Our aim was to select strains that produce the highest quantities of H(2)S as the main product of L-cysteine catabolism. Thirty seven yeast and bacteria strains were cultivated with or without L-cysteine. The separation between the growth medium and the H(2)S trapping layer displayed good performance: all the studied strains grew efficiently and only negligible loss of H(2)S was observed during culturing. The strains displayed large differences in their H(2)S production capabilities: yeast strains were greater producers of H(2)S than bacteria with production strain-related in both cases. Furthermore, the relationship between H(2)S production and L-cysteine consumption was analyzed, which made it possible for us to select microorganisms with high capacity in L-cysteine degradation. The production of volatile sulfur compounds was also studied and the possible effect of culture pH and metabolic differences between strains are discussed.     

Selective isolation and study on the global distribution of the genus Planobispora in soils.

Planobispora strains, including the type strain, produced abundant sporangia on humic acid-vitamin gellan gum medium when more than 50 colonies grew on a plate. However, these strains did not produce sporangia on the aerial mycelium, a diagnostic characteristic of this genus, when fewer than 20 colonies grew on a plate. Trace salts, such as FeSO4 x 7H2O, MnCl2 x 4H2O, ZnSO4 x 7H2O, and NiSO4 x 6H2O, stimulated sporangium formation of Planobispora strains. Better sporangium formation of Planobispora strains was observed on the humic acid-trace salts gellan gum medium at pH 9.0 than at neutral pH. Moreover, an alkaline medium repressed the growth of three out of six Streptomyces strains so that this condition was effective for selective isolation of Planobispora strains. An alkaline flooding solution of skim milk, five antimicrobial agents, and dry heat treatment at 90 degrees C for 60 min were effective for selective isolation. Using these techniques, we examined the distribution of Planobispora strains by using 1467 soil samples collected from Japan and other parts of the world. One hundred and nineteen Planobispora strains were isolated from 51 soil samples (3.5% of the samples tested) that were collected in Ecuador, Egypt, French Guiana, India, and Madagascar. Planobispora strains were recovered only in tropical and subtropical soils. To our knowledge, this is the first record that Planobispora strains have been isolated from locations other than Venezuela, Namibia, or South Africa.     

Response surface methodology for optimizing the fermentation medium of alpha-galactosidase in solid-state fermentation

AIMS: Alpha-galactosidase is applied in food and feed industries for hydrolysing raffinose series oligosaccharides (RO) that are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. The objective of the current work was to develop an optimal culture medium for the production of alpha-galactosidase in solid-state fermentation (SSF) by a mutant strain Aspergillus foetidus. METHODS AND RESULTS: Response surface methodology (RSM) was applied to evaluate the effects of variables, namely the concentrations of wheat bran, soybean meal, KH(2)PO(4), MnSO(4).H(2)O and CuSO(4).5H(2)O on alpha-galactosidase production in the solid substrate. A fractional factorial design (FFD) was firstly used to isolate the main factors that affected the production of alpha-galactosidase and the central composite experimental design (CCD) was then adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was composed of 8.2137 g wheat bran, 1.7843 g soybean meal, 0.001 g MnSO(4).H(2)O and 0.001 g CuSO(4).5H(2)O in 10 g dry matter fermentation medium. CONCLUSIONS: After incubating 96 h in the optimum fermentation medium, alpha-galactosidase activity was predicted to be 2210.76 U g(-1) dry matter in 250 ml shake flask. In the present study, alpha-galactosidase activity reached 2207.19 U g(-1) dry matter. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimization of the solid substrate was a very important measure to increase enzyme activity and realize industrial production of alpha-galactosidase. The process of alpha-galactosidase production in laboratory scale may have the potential to scale-up.     

A proton-translocating ATPase regulates pH of the bacterial cytoplasm

Regulatory mechanisms of cytoplasmic pH in Streptococcus faecalis with no respiratory chain were investigated. In a mutant defective in cytoplasmic alkalization conducted by a proton-translocating ATPase (H+-ATPase), the cytoplasmic pH is approximately 0.4 to 0.5 pH units lower than the medium pH, at pH 5.5 to 9.0. The cytoplasmic pH of the wild-type strain was always higher than that of the mutant at a pH below 8 and was the same as that of the mutant at an alkaline pH over 8. Thus, the cytoplasmic pH is regulated only by the cytoplasmic alkalization, and there is no regulation at alkaline pH in S. faecalis. A generation of the protonmotive force conducted by the H+-ATPase depended on the cytoplasmic pH rather than the medium pH, and the generation decreased rapidly when the cytoplasmic pH was increased over 7.7. The decrease at alkaline pH was not caused by increases in the rate of proton influx. These results suggest that cytoplasmic alkalization is diminished when alkaline pH of the cytoplasm is over 7.7, because of a low activity of proton extrusion by the H+-ATPase, and consequently, the cytoplasmic pH is regulated at about 7.7. The cytoplasmic pH was regulated at a high level in cells that had a high level of H+-ATPase. I conclude that in S. faecalis, the cytoplasmic pH is regulated by H+-ATPase.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

pH effects on 10 Streptomyces spp. growth and sporulation depend on nutrients

AIMS: Streptomycetes are regarded to prefer neutral to alkaline environmental pH, although they commonly occur at remarkably variable pH and nutritional conditions. Therefore, the dependence of 10 Streptomyces spp. pH tolerance on nutrients was determined. METHODS AND RESULTS: Ten environmental Streptomyces spp. were grown and sporulated between pH 4.0 and 11.5, at the interval of 1.5, on starch-casein-KNO(3), tryptone-yeast extract-glucose, glycerol-arginine and tryptone-soy agars, and three their modifications. On media with starch and casein; glucose, tryptone and yeast extract; tryptone and soy peptone; and glycerol-arginine and yeast extract strains grew over a broad pH range between 4.0-5.5 and 10.0-11.5. On glycerol-arginine and on medium with Na-propionate, NH(4)NO(3) and yeast extract, streptomycetes grew optimally at pH 7.0 and above. The high organic load enabled the growth over a wide pH range. The sporulation pH ranges followed those for growth. CONCLUSIONS: The high organic load enabled the growth over a wide pH range. The strain-specific differences in sporulation were greater than those caused by pH. The best medium for sporulation contained glucose and tryptone with minerals of glycerol-arginine agar at pH 5.5. SIGNIFICANCE AND IMPACT OF THE STUDY: The growth pH ranges, pH ranges for the optimal growth, and sporulation were strongly dependent on nutrients.     

Adaptive responses of Salmonella enterica serovar Typhimurium DT104 and other S. Typhimurium strains and Escherichia coli O157 to low pH environments

AIMS: Cattle are a known main reservoir for acid-resistant Escherichia coli O157 and Salmonella enterica serovar Typhimurium DT104. We studied the response of S. Typhimurium DT104 to extreme low pH environments and compared their response to that of acid-resistant E. coli O157 and other S. Typhimurium phage types. METHODS AND RESULTS: Bacteria were grown in nutrient-rich medium and subsequently acid challenged at pH 2.5. We found that stationary phase cultures of various S. Typhimurium strains were able to survive a challenge for 2 h at pH 2.5. As in E. coli, the ability of S. Typhimurium to survive at pH 2.5 was shown to be dependent on the presence of amino acids, specifically arginine. The amount of proton pumping H+/ATPase, both in E. coli O157 and S. Typhimurium strains, was lower when grown at pH values <6 than after growth at pH 7.5. Cyclo fatty acid content of membranes of bacteria grown at pH values <6 was higher than that of membranes of bacteria grown at pH 7.5. CONCLUSIONS: Various S. Typhimurium strains, both DT104 and non-DT104, are able to survive for a prolonged period of time at pH 2.5. Their response to such low pH environment is seemingly similar to that of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-borne pathogens like S. Typhimurium DT104 and E. coli O157 form a serious threat to public health since such strains are able to survive under extreme low pH conditions as present in the human stomach. The emergence these acid-resistant strains suggests the presence of a selection barrier. The intestinal tract of ruminants fed a carbohydrate-rich diet might be such a barrier.     

Isolation,identification and some cultural conditionsof a protease-producing thermophilic Streptomyces straingrown on chicken feather as a substrate

Six strains of thermophilic actinomycetes were isolated from soil using an enrichmenttechnique with feathers as the sole carbon and nitrogen source. They showed clear proteolyticactivity on casein agar medium. The most active strain was tentatively identified as Streptomycesthermonitrificans. This isolate was grown in a basal medium with feathers and:or other carbon andnitrogen sources. Supernatant from centrifuged cultures was examined for protease activity andtemperature and pH optima were determined for enzyme activity. Optimum proteolytic activity onbasal liquid medium containing 1% chicken feather pieces was obtained at 50°C, in a mediumadjusted at pH8 and incubated for 72 h at 150 rpm. Proteolytic activity was further increased by1.5% feather pieces and the time required for maximal activity was 96 h. The keratinolytic activityof S. thermonitrificans was examined by incubation with native chicken feather pieces and it wasfound that it is significantly active. The degradation of whole intact feathers by S.thermonitrificans was obtained after 48 h of incubation at 50°C. The pH and temperature optimafor proteolytic activity were 9.0 and 50°C, respectively. The proteolytic activity was stable at40°C for 1 h. The proteolytic activity was inhibited by DFP but not by EDTA or pCMB. Theseresults inidicated that the enzyme(s) can be classified as an alkaline protease. 1999 ElsevierScience Ltd. All rights reserved.     

Acid resistance variability among isolates of Salmonella enterica serovar Typhimurium DT104

AIMS: Acid resistance could be an indicator of virulence since acid resistant strains are able to better survive the human stomach passage and in macrophages. We studied the acid resistance of several Salmonella Typhimurium DT104 strains isolated from food and humans and identified cellular parameters contributing to the enhanced acid resistance of these isolates. METHODS AND RESULTS: Acid resistance was tested in 37 Salmonella enterica Typhimurium serovar DT104 (S. Typhimurium DT104) strains. Acid adaptation at pH 5 followed by exposure for 2 h at pH 2.5 in the 27 human, nine nonhuman, and in two reference strains, revealed strong variation of acid survival. After 2 h at pH 2.5 six strains of S. Typhimurium DT104 were considered high acid resistant as they displayed a level of survival >10%, 14 strains were considered intermediate acid resistant (level of survival was <10% and >0.01%) and 19 strains were considered low acid resistant (level of survival <0.01%). Six strains were selected for further studies and proteomics revealed a relatively high amount of phase 2 flagellin in an acid-sensitive strain and a relatively high amount of the beta component of the H(+)/ATPase in an acid-resistant strain. Two strains were slightly more heat resistant possibly as the result of increased levels of DnaK or GroEL. CONCLUSIONS: A significant difference could be detected between human and food isolates regarding their acid resistance; all high acid-resistant strains were human isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: S. Typhimurium DT104 is known for two decades and has a great impact on human health causing serious food-borne diseases. Our results suggest the existence of a positive correlation between acid resistance and pathogenicity in S. Typhimurium DT104 as all high acid-resistant strains were isolated from humans.     

Adaptation of a neutrophilic dairy-associated Bacillus cereus isolate to alkaline pH

AIMS: This study identified and studied the response of five Bacillus strains, isolated from alkaline cleaning in place (CIP) solutions, to alkaline conditions. METHODS AND RESULTS: Isolates were identified as B. cereus by 16S rDNA sequencing. External and internal cell pH and buffering capacity data of a representative strain, Bacillus DL5, were compared to B. cereus ATCC 10702. Results indicated that a buffering system was induced when the pH of the growth medium increased to above pH 10, which was effective up to pH 12 and presumably cell wall associated. Volume measurements and confocal scanning laser microscope images of Bacillus DL5 cells showed that cells exhibited more pronounced stress symptoms when exposed to pH 10 than at pHs above 10. Long-term exposure of Bacillus DL5 to pH 10 or 10.5 indicated that cells grew in planktonic form and formed biofilms at both pHs. CONCLUSIONS: Bacillus DL5 was a neutrophile with a growth pH range similar to B. cereus ATCC 10702, but tolerated alkaline pH. This may be a general trait of the B. cereus species rather than a specific phenomenon of isolates from alkaline ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Other neutrophilic B. cereus isolates may exhibit similar responses to alkaline conditions as the isolates studied here. These results may have important implications for dairy manufacturers.     

Two-Tube Test for the Rapid Identification of Prompt Lactose-Fermenting Genera Within the Family Enterobacteriaceae

One hundred and fifty-nine prompt lactose-fermenting strains of Enterobacteriaceae were tested for H(2)S production, ornithine decarboxylase activity, motility, and citrate utilization by using two different sets of test media. It was shown that the three tests first mentioned could be carried out in a single tube, the results being as reliable as when each was done separately. Simmons medium was preferred for the detection of citrate utilization. Since these four tests require only two tubes, they are considered more convenient in routine diagnostics. A combined medium for the detection of H(2)S production and motility is described.     

Acidophilic haloarchaeal strains are isolated from various solar salts

Haloarchaeal strains require high concentrations of NaCl for their growth, with optimum concentrations of 10–30%. They display a wide variety of morphology and physiology including pH range for growth. Many strains grow at neutral to slightly alkaline pH, and some only at alkaline pH. However, no strain has been reported to grow only in acidic pH conditions within the family Halobacteriaceae. In this study, we isolated many halophiles capable of growth in a 20% NaCl medium adjusted to pH 4.5 from 28 commercially available salts. They showed growth at pH 4.0 to 6.5, depending slightly on the magnesium content. The most acidophilic strain MH1-52-1 isolated from an imported solar salt (pH of saturated solution was 9.0) was non-pigmented and extremely halophilic. It was only capable of growing at pH 4.2–4.8 with an optimum at pH 4.4 in a medium with 0.1% magnesium chloride, and at pH 4.0–6.0 (optimum at pH 4.0) in a medium with 5.0% magnesium. The 16S rRNA and DNA-dependent RNA polymerase subunit B' gene sequences demonstrated clearly that the strain MH1-52-1 represents a new genus in the family Halobacteriaceae.     

Production of Shiga toxin by Shiga toxin-expressing Escherichia coli (STEC) in broth media: from divergence to definition

AIMS: To determine the suitability of eight different commercial broth media for Shiga toxin (Stx) production. METHODS AND RESULTS: Shiga toxin-producing Escherichia coli (STEC) strains producing Stx1 or Stx2 were grown at 37 degrees C (250 rev min(-1)) for 24 h in brain heart infusion broth, E. coli broth, Evans medium, Luria-Bertani broth, Penassay broth, buffered-peptone water, syncase broth and trypticase soy broth. Toxin production was measured by enzyme-linked immunosorbent assay (ELISA) in polymyxin-treated cell pellets and/or supernatants of cultures, ELISA optical densities reached 1 when isolates were grown for 2-4 h in E. coli broth in the presence of antibiotic. Besides, a collection of STEC-expressing Stx strains was evaluated and the Stx production was assayed in the supernatants and in polymyxin-treated pellets of bacterial growth after 4 h of cultivation in E. coli broth in the presence of antibiotic. CONCLUSIONS: The most suitable medium for Stx production was E. coli broth when the bacterial isolates were grown for 4 h in the presence of ciprofloxacin and the Stx production is detected in the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first comprehensive comparison of different broth media with regard to Stx production to establish optimal culture conditions for STEC detection in routine diagnostic laboratories.     

Fermentation conditions affecting the bacterial growth and exopolysaccharide production by Streptococcus thermophilus ST 111 in milk-based medium

AIMS: To study the effect of different fermentation conditions and to model the effect of temperature and pH on different biokinetic parameters of bacterial growth and exopolysaccharides (EPS) production of Streptococcus thermophilus ST 111 in milk-based medium. METHODS AND RESULTS: The influence of temperature and pH was studied through fermentation and modelling. Fermentations under non-pH controlled conditions with S. thermophilus ST 111 indicated that the EPS production was low in milk medium, even if additional nitrogen sources were supplemented. Under pH-controlled conditions, addition of whey protein hydrolysate to the milk medium resulted in a fivefold increase of the EPS production. This medium did not contain polysaccharides interfering with EPS isolation. Primary and secondary modelling of different fermentations revealed an optimum temperature and pH of 40 degrees C and constant pH 6.2, respectively, for growth in milk medium supplemented with whey protein hydrolysate. Maximum EPS production was observed in the range of 32-42 degrees C and constant pH 5.5-6.6. Whereas growth and maximum EPS production were clearly influenced by temperature and pH, the specific EPS production was only affected by stress conditions (T = 49 degrees C). CONCLUSIONS: Addition of whey protein hydrolysate to milk medium resulted in an increased growth and EPS production of S. thermophilus ST 111 under pH-controlled conditions. A modelling approach allowed studying the influence of temperature and pH on the kinetics of both growth and EPS production. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of an appropriate milk-based medium and a combined model of temperature and pH can be of practical importance for the production of yoghurt or other fermented milks as well as for process optimization of the large-scale production of starter strains to be used for their EPS production.