Guttation droplets of <Emphasis Type="Italic">Penicillium nordicum</Emphasis> and <Emphasis Type="Italic">Penicillium verrucosum</Emphasis> contain high concentrations of the mycotoxins ochratoxin A and B

Eight of eleven ochratoxigenic isolates of Penicillium nordicum and Penicillium verrucosum produced guttation droplets when grown on Czapek yeast extract (CYA) agar for 10–14 days at 25°C. Parallel cultivation of one strain each of P. nordicum and P. verrucosum on malt extract agar demonstrated that higher volumes of exudate are produced on this agar. However, HPLC analyses revealed higher concentrations of ochratoxin A (OTA) and B (OTB) in droplets originating from cultures on CYA. For quantitative determination of the mycotoxin contents, triplicates of three isolates each of P. nordicum and P. verrucosum were grown as single spot cultures on CYA for up to 14 days at 25°C. Guttation droplets were carefully collected between day 11 and 14 with a microliter syringe from each culture. Extracts from exudates and corresponding mycelia as well as fungal free agar were analyzed by HPLC for the occurrence of ochratoxin A (OTA) and B (OTB). Mean concentrations ranging between 92.7–8667.0 ng OTA and 159.7–2943.3 ng OTB per ml were detected in the guttation fluids. Considerably lower toxin levels were found in corresponding samples of the underlying mycelia (9.0–819.3 ng OTA and 4.5–409.7 ng OTB/g) and fungal free agar (15.3–417.0 ng OTA and 12.7–151.3 ng OTB/g). This is the first report which shows that high amounts of mycotoxins could be excreted from toxigenic Penicillium isolates into guttation droplets.     

Biotechnological process for obtaining new fermented products from cashew apple fruit by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains

In Brazil, the use of cashew apple (Anacardium occidentale L.) to obtain new products by biotechnological process represents an important alternative to avoid wastage of a large quantity of this fruit, which reaches about 85% of the annual production of 1 million tons. This work focuses on the development of an alcoholic product obtained by the fermentation of cashew apple juice. The inoculation with two different strains of yeast Saccharomyces cerevisiae viz. SCP and SCT, were standardized to a concentration of 10⁷ cells ml⁻¹. Each inoculum was added to 1,500 ml of cashew must. Fermentation was performed at 28 ± 3°C and aliquots were withdrawn every 24 h to monitor soluble sugar concentrations, pH, and dry matter contents. The volatile compounds in fermented products were analyzed using the gas chromatography/mass spectrometry (GC/MS) system. After 6 days, the fermentation process was completed, cells removed by filtration and centrifugation, and the products were stabilized under refrigeration for a period of 20 days. The stabilized products were stored in glass bottles and pasteurized at 60 ± 5°C/30 min. Both fermented products contained ethanol concentration above 6% (v v⁻¹) while methanol was not detected and total acidity was below 90 mEq l⁻¹, representing a pH of 3.8–3.9. The volatile compounds were characterized by the presence of aldehyde (butyl aldehyde diethyl acetal, 2,4-dimethyl-hepta-2,4-dienal, and 2-methyl-2-pentenal) and ester (ethyl α-methylbutyrate) representing fruity aroma. The strain SCT was found to be better and efficient and this produced 10% more alcohol over that of strain SCP.     

Salinity tolerance of aRhizobium meliloti strain isolated from salt affected soils

The salinity tolerance of aRhizobium meliloti strain isolated from salt-affected soils was examined. Growth of the strain on yeast—mannitol broth containing 0–1.2% NaCl exhibited in all cases the same generation time and simultaneous onset of the stationary phase while the total viable number of cells was the same for three continuous generations. The nodulation, plant yield and elemental composition ofMedicago sativa plants grown on agar slopes, inoculated with cultures from the third generation grown on broth containing 0–1.2% NaCl responded identically to all inocula. The salinity tolerance of the strain in fixing nitrogen was furthermore demonstrated withM. sativa plants grown on either nitrogen-free agar slopes containing 0.2–1.2% NaCl or soil-agar slopes with saline soil in which 0.15 and 0.3% additional NaCl was used.     

<Emphasis Type="Italic">Aphanomyces sinensis</Emphasis> sp. nov., isolated from juvenile soft-shelled turtle, <Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>, in Japan

A species of Aphanomyces was isolated from juvenile soft-shelled turtles, Pelodiscus sinensis, cultured in Japan. Typically, an infected turtle showed small whitish maculae on the carapace. Many hyphae were observed in the epidermis. The hyphae were isolated using glucose–yeast (GY) agar plates. The morphological characteristics were very similar to those of Aphanomyces laevis, but a clear nuclear spot was observed in the center of the oospore in the strains isolated from the soft-shelled turtles. The optimal growth temperature for the isolates was 25–30°C and the optimum pH was 6–9. Experimental infection tests with isolates produced small whitish maculae on the carapace, and soft-shelled turtles artificially infected with the zoospores showed high mortality, especially in the high-dose group. Phylogenetic analysis based on the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) indicated that the isolates from the soft-shelled turtles were unidentified species of Aphanomyces. As a result, the strain was described as a new species, Aphanomyces sinensis.     

Enhancement of fruity aroma production of Pseudomonas fragi grown on skim milk,whey and whey permeate supplemented with C3-C7 fatty acids

Summary Pseudomonas fragi strain CRDA 037 produced a fruity aroma when grown in skim milk-, whey-and whey permeate-based culture media. The production of the odour-active metabolites was related to the lipid content of these media but was not influenced by the pH of the cultures. Analysis of the fruity aroma revealed that esters of fatty acids were some of the odouractive metabolites. Addition of C₃-C₇ fatty acids to the culture at 0 h stimulated the production of the corresponding fatty acid esters from 12 to 1570 times compared to unsupplemented media. Supplementation of the culture media with the C₃-C₇ fatty acids at 48 h, resulted in a 1.4- to 932-fold increase in the ethyl ester concentration.     

Decalactone Production by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> under increased O<Subscript>2</Subscript> Transfer Rates

Yarrowia lipolytica converts methyl ricinoleate to γ-decalactone, a high-value fruity aroma compound. The highest amount of 3-hydroxy-γ-decalactone produced by the yeast (263 mg l^-1) occurred by increasing the k_La up to 120 h⁻¹ at atmospheric pressure; above it, its concentration decreased, suggesting a predominance of the activity of 3-hydroxyacyl-CoA dehydrogenase. Cultures were grown under high-pressure, i.e., under increased O₂ solubility, but, although growth was accelerated, γ-decalactone production decreased. However, by applying 0.5 MPa during growth and biotransformation gave increased concentrations of dec−2-en-4-olide and dec-3-en-4-olide (70 mg l⁻¹).     

Isolation and Characterization of Antifungal Substances from Burkholderia sp. Culture Broth

A new antagonistic Burkholderia strain, designated MP-1 and producing antifungal activities against various filamentous plant pathogenic fungi, was isolated from the rhizoshere in the Naju area. Cultural characteristic studies strongly suggested that this strain belongs to the genus Burkholderia. The nucleotide sequence of the 16S rRNA gene (1491 pb) of strain MP-1 exhibited close similarity (99% to 100%) with other Burkholderia 16S rRNA genes. Extraction of fermentation broth of Burkholderia sp. MP-1 and various separations and purification steps led to isolation of four pure active molecules. The chemical structure of these four compounds—named phenylacetic acid, hydrocinnamic acid, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetate methyl ester—was established on the basis on their gas chromatography–electron impact–mass spectrometry (GC-EI-MS) and trimethylsilation GC-EI-MS data. The four isolated compounds inhibited filamentous fungal growth on potato dextrose agar medium supplemented with 100 mg/L of phenylacetic acid, hydrocinnamic acid, 4-hydroxyphenylacetic acid and 4-hydroxyphenylacetate methyl ester individually.     

Growth and mycotoxin production by <Emphasis Type="Italic">Chaetomium globosum</Emphasis>

Chaetomium globosum, the most common species within this genus, produces chaetoglobosins A and C when cultured on building material. Relatively low levels of these compounds have been shown to be lethal to various tissue culture cell lines. This study had two major objectives: (1) to determine the frequency at which Chaetomium species are isolated in water-damaged buildings and (2) to examine the production of chaetoglobosins A and C in isolates of C. globosum obtained from different buildings. Out of 794 water-damaged buildings, Chaetomium species were isolated in 49% of these structures. C. globosum ATCC 16021 was grown on four different media: oatmeal agar (OA), potato dextrose agar (PDA), corn meal agar (CMA), and malt extract agar (MEA). After 4 weeks, fungal growth was evaluated based on colony diameter and the quantity of spores produced on agar plates. In addition, production of chaetoglobosin A and C was monitored using high performance liquid chromatography. Colony diameter, spore production, and mycotoxin production by C. globosum were the highest on OA. Out of 30 C. globosum isolates cultured on OA for 4 weeks, 16 produced detectable amounts of chaetoglobosin A and every isolate produced chaetoglobosin C.     

The production of hydrogen sulphide and other aroma compounds by wine strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in synthetic media with different nitrogen concentrations

The aim of this study was to evaluate the combined effect of initial nitrogen content on the production of hydrogen sulphide and other volatile compounds during alcoholic fermentation. For that propose, three commercial wine strains of Saccharomyces cerevisiae were used to ferment synthetic grape juice media under different nitrogen concentrations. H₂S was measured throughout fermentations and other aroma compounds were analyzed at the end of the experiments. The trigger levels at which an inverse relationship between the initial nitrogen present in media and total H₂S production varied among the three strains tested. For UCD522 and PYCC4072, the highest H₂S levels were produced in media with 267 mg N l⁻¹ of initial nitrogen, whereas the lowest levels were detected with nitrogen limitation/starvation conditions (66 mg N l⁻¹). Moreover, 21 other aroma compounds belonging to different chemical classes were identified and quantified by solid phase micro-extraction (SPME) coupled to gas chromatography–mass spectrometry (GC–MS). The initial nitrogen concentration more than yeast strain had a decisive effect on the final aroma composition, suggesting that modulation of nutrients emerges as a useful tool for producing desired flavour and odour compounds.     

<Emphasis Type="Italic">Arthrobacter halodurans</Emphasis> sp. nov., a new halotolerant bacterium isolated from sea water

A novel Gram-positive, halotolerant, non-sporulating, non-motile, catalase-positive, oxidase-negative and aerobic bacterium, designated strain JSM 078085^T, was isolated from sea water collected from the South China Sea. Strain JSM 078085^T exhibited a rod-coccus growth cycle and produced a yellow pigment. The strain was able to grow in the presence of 0–12% (w/v) NaCl and at pH 6.0–9.5 and 4–35°C; optimum growth was observed at pH 7.0 and 25–30°C in the absence of NaCl. The peptidoglycan type was A4α (l-Lys–l-Ala–l-Glu). Cell-wall sugars contained galactose and glucose. Strain JSM 078085^T contained menaquinone MK-9(H₂) as the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. The major cellular fatty acids were anteiso-C_15:0, iso-C_15:0 and anteiso-C_17:0 and the DNA G + C content was 63.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 078085^T should be assigned to the genus Arthrobacter, being most closely related to the type strain of Arthrobacter rhombi (sequence similarity 97.1%), and the two strains formed a distinct lineage in the phylogenetic tree. The level of DNA–DNA relatedness between strain JSM 078085^T and the type strain of Arthrobacter rhombi was 10.6%. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 078085^T represents a novel species of the genus Arthrobacter, for which the name Arthrobacter halodurans sp. nov. is proposed. The type strain is JSM 078085^T (=DSM 21081^T=KCTC 19430^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 078085^T is EU583729.     

One-step transformation of the dimorphic yeast Yarrowia lipolytica

An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 10⁵ transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000 concentration and the wetness of selective plates were investigated. Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997     

Extracellular β-agarase LSL-1 producing neoagarobiose from a newly isolated agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

Summary An agar-liquefying Acinetobacter species capable of utilizing agar as sole source of carbon and energy was isolated from soil samples and the culture conditions were standardized for the maximal production of extracellular agarase. The bacterium was capable of liquefying an agar-plate within 3 days of incubation and produced extracellular agarase within a short period of time (16–18 h) when grown in defined mineral salts medium. Bacterium grew in the pH range 4.0–9.0, optimal at pH 7.0; temperature 25–40 °C and optimal at 37 °C. The agarase secreted by the Acinetobacter strain was inducible by agar and not repressed by other simple sugars when supplemented along with agar in the medium. The bacterium did not require NaCl for growth or production of agarase. The bacterium did not utilize other polysaccharides like κ-carrageenan, alginate, cellulose, and CMC. The activity staining of partially purified agarase preparations after native-PAGE and SDS PAGE revealed the presence of a single zone of clearance corresponding to the molecular weight 100 kDa, suggesting that it is a monomer. Neoagarobiose was the end product of agarose hydrolysis by this enzyme. The agarase was an endo-type glycosidase and belongs to Group-III β-agarase family.     

Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation

The use of inert absorbent polymeric supports for cellular attachment in solid-state fungal fermentation influenced growth, morphology, and production of bioactive secondary metabolites. Two filamentous fungi exemplified the utility of this approach to facilitate the discovery of new antimicrobial compounds. Cylindrocarpon sp. LL-Cyan426 produced pyrrocidines A and B and Acremonium sp. LL-Cyan416 produced acremonidins A–E when grown on agar bearing moist polyester–cellulose paper and generated distinctly different metabolite profiles than the conventional shaken or stationary liquid fermentations. Differences were also apparent when tenfold concentrated methanol extracts from these fermentations were tested against antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria, and zones of inhibition were compared. Shaken broth cultures of Acremonium sp. or Cylindrocarpon sp. showed complex HPLC patterns, lower levels of target compounds, and high levels of unwanted compounds and medium components, while agar/solid support cultures showed significantly increased yields of pyrrocidines A and B and acremonidins A–E, respectively. This method, mixed-phase fermentation (fermentation with an inert solid support bearing liquid medium), exploited the increase in surface area available for fungal growth on the supports and the tendency of some microorganisms to adhere to solid surfaces, possibly mimicking their natural growth habits. The production of dimeric anthraquinones by Penicillium sp. LL-WF159 was investigated in liquid fermentation using various inert polymeric immobilization supports composed of polypropylene, polypropylene cellulose, polyester–cellulose, or polyurethane. This culture produced rugulosin, skyrin, flavomannin, and a new bisanthracene, WF159-A, after fermentation in the presence and absence of polymeric supports for mycelial attachment. The physical nature of the different support systems influenced culture morphology and relative metabolite yields, as determined by HPLC analysis and measurement of antimicrobial activity. The application of such immobilized-cell fermentation methods under solid and liquid conditions facilitated the discovery of new antibiotic compounds, and offers new approaches to fungal fermentation for natural product discovery.     

Morphological,Physiological, and Molecular Characterization of a Newly Isolated Steroid-Degrading Actinomycete,Identified as <Emphasis Type="Italic">Rhodococcus ruber</Emphasis> Strain Chol-4

The aerobic degradation of cholesterol, testosterone, androsterone, progesterone, and further steroid compounds as sole carbon source has been observed in the newly isolated bacterial Gram-positive strain Chol-4. The 16S rRNA gene sequence shares the greatest similarity with members of the genus Rhodococcus, with the closest shared nucleotide identities of 98–99% with Rhodococcus ruber (DSM 43338^T) and Rhodococcus aetherivorans (DSM 44752^T). Phylogenetic analysis of Rhodococcus 16S rRNA gene sequences consistently places strain Chol-4 in a clade shared with those both type strains within the Rhodococcus rhodochrous subclade. The results of DNA–DNA hybridization against its two phylogenetically closest neighbors as well as the results of morphological, physiological, and biochemical tests allowed genotypic and phenotypic differentiation of strain Chol-4 from Rhodococcus ruber (DSM 43338^T) on the species level and from the other validly described Rhodococcus species on the genus level. Strain Chol-4 therefore merits recognition as a novel strain of the species Rhodococcus ruber and demonstrates for the first time the capability of this species to utilize a great variety of steroid compounds as growth substrates never shown for other species of this genus so far. The genome of strain Chol-4 harbors at least one gene cluster that may be responsible for the degradation of steroid compounds. This gene cluster was identified in a cloned 5458 bp BamHI–EcoRV DNA fragment and compared to similar genes from other Gram-positive and Gram-negative bacteria described so far.     

A novel extracellular phospholipase C purified from a marine bacterium, <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> sp. J937

Marine bacterial isolates were screened for phospholipase C (PLC) activity on PCY agar plates containing phosphatidylcholine (PC) as substrate. The strain that showed the highest activity on a PCY screening agar plate and a thin-layer chromatography was identified as a strain of Pseudoalteromonas and subsequently designated Pseudoalteromonas sp. J937. The extracellular PLC of the strain J937 was purified to a specific activity of 33 U mg⁻¹ protein by serial ion exchange and gel filtration column chromatography. It had a molecular mass of 32 kDa estimated by SDS–PAGE. The optimal pH and temperature of the enzyme were about pH 8 and 45°C, respectively. The PLC hydrolyzed phosphatidylethanolamine as well as PC but not other glycerophospholipids. Its activity was enhanced by 150% with Ca²⁺ (200 mM) and by 180% with Na⁺ (500 mM), suggesting that the purified PLC is a marine-type enzyme.     

<Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain XTBG34 promotes plant growth by producing 2-pentylfuran

Several chemical changes in soil are associated with plant growth-promoting rhizobacteria. An endosporeforming bacterium, strain XTBG34, was isolated from a Xishuangbanna Tropical Botanical Garden soil sample and identified as Bacillus megaterium. The strain’s volatiles had remarkable plant growth promotion activity in Arabidopsis thaliana plants; after 15 days treatment, the fresh weight of plants inoculated with XTBG34 was almost 2-fold compared with those inoculated with DH5α. Head space volatile compounds produced by XTBG34, trapped with headspace solid phase microextraction and identified by gas chromatography-mass spectrometry, included aldehydes, alkanes, ketones and aroma components. Of the 11 compounds assayed for plant growth promotion activity in divided Petri plates, only 2-pentylfuran increased plant growth. We have therefore identified a new plant growth promotion volatile of B. megaterium XTBG34, which deserves further study in the mechanisms of interaction between plant growth-promoting rhizobacteria and plants.     

Isolation of mutants of Penicillium purpurogenum resistant to catabolite repression

 The strain Penicillium purpurogenum P-26 was subjected to UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine treatment and mutants were isolated capable of synthesizing cellulase under the conditions of a high concentration of glucose. Initially mutants resistant to catabolite repression by 2-deoxy-D-glucose were isolated on Walseth’s cellulose/agar plates containing 15–45 mM 2-deoxy-D-glucose. These mutants were again screened for resistance to catabolite repression by glycerol or glucose on Walseth’s cellulose/agar plates containing 50 g/l glycerol or 50 g/l glucose respectively. Four mutants with different sizes of clearing zone on Walseth’s cellulose/agar plates containing 50 g/l glucose were selected for flask culture. Among them, the mutant NTUV-45-4 showed better carboxymethylcellulase activity in flask culture containing 1% Avicel plus 3% glucose than did the parental strain. Received: 9 October 1995/Received revision: 27 November 1995/Accepted: 8 January 1996     

Restoration of culturability of starvation-stressed and low-temperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds

Late-exponential-phase cells of Escherichia coli O157:H- strain E32511/HSC became nonculturable in sterilized distilled water microcosms at 4 °C. Plate counts declined from 3 × 10⁶ to less than 0.1 CFU/ml in about 21 days. However, when samples of microcosms at 21 days were inoculated onto an agar medium amended with catalase or nonenzyme peroxide-degrading compounds such as sodium pyruvate or α-ketoglutaric acid, plate counts increased to 10⁴–10⁵ CFU/ml within 48 h. The proposed mode of action of the catalase or pyruvate is via the degradation of the metabolic by-product H₂O₂, rather than through supplementation of a required nutrient in the recovery of nonculturable cells. Our studies were based on the assumption that E32511/HSC strain responds to starvation and a low temperature by entering a nonculturable state and that the correction of oxidative stress upon the inoculation of bacteria on agar plates promotes recovery of nonculturable cells. Received: 15 January 1999 / Accepted: 8 April 1999     

Optimization of the production of aroma compounds by Kluyveromyces marxianus in solid-state fermentation using factorial design and response surface methodology

Studies were carried out for the production of aroma compounds in solid-state fermentation using factorial design and response surface methodology (RSM) experiments. Five agro-industrial residues were evaluated as substrate for cultivating a strain of Kluyveromyces marxianus. The results proved the feasibility of using cassava bagasse and giant palm bran (Opuntia ficus indica) as substrates to produce fruity aroma compounds by the yeast culture. In order to test the influence of the process parameters on the culture to produce volatile compounds, two statistical experimental designs were performed. The parameters studied were initial substrate pH, addition of glucose, cultivation temperature, initial substrate moisture and inoculum size. Using a 2(5) factorial design, addition of glucose and initial pH of the substrate was found statistically significant for aroma compounds production on palm bran. Although this experimental design showed that addition of glucose did not have a significant role with cassava bagasse, 2(2) factorial design revealed that glucose addition was significant at higher concentrations. Head-space analysis of the culture by gas chromatography showed the production of nine and eleven compounds from palm bran and cassava bagasse, respectively, which included alcohols, esters and aldehyde. In both the cases, two compounds remained unidentified and ethyl acetate, ethanol and acetaldehyde were the major compounds produced. Esters produced were responsible for the fruity aroma in both the cases. With palm bran, ethanol was the compound produced in highest concentration, and with cassava bagasse (both supplemented with 10% glucose), ethyl acetate was produced at highest concentration, accumulating 418 and 1395μmoll(-1) head-spaceg(-1) substrate in 72h, respectively.     

Fruiting ofLyophyllum tylicolor in plate culture on Soytoneglucose agar and urea-treated soil extract agar

Lyophyllum tylicolor, which forms mycelial basidia (and basidiospores), produced fruit-bodies when cultivated at 20°C under continuous illumination of 400–700 lux on agar plates containing Bacto-Soytone and glucose or an extract from urea-treated soil. Under these conditions, mycelial basidia were also observed on the Soytone-glucose agar, but not on the soil extract agar. In darkness, fruit-bodies and mycelial basidia were not observed on either medium. In culture on the soil extract agar, fruit-body primordia were produced at the position of the margin of the colony when it was transferred from darkness to continuous light; stipes did not elongate under illumination of ca. 2000 lux; and mycelial basidia and basidiospores, but not fruit-bodies, developed when glucose concentration in the medium was as high as 1% (w/v).