Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium.

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

Measurement of yeast concentrations in liquid hydrocarbon fermentations: influence of experimental conditions and statistical significance of the results obtained

The influence of the following factors on the measurement of yeast concentration in liquid hydrocarbon fermentations was studied: surfactant, type (Tween 20, Tween 80, and G 3300), yeast concentration (0.215 to 0.272 and 3.37 to 4.08 g/liter, as dry matter), oil concentration (8.32 and 61.6 g/liter), surfactant, concentration (0.515, 1.030, 1.545, and 2.060 g/liter), and time of contact of the surfactant with the oil-yeast-aqueous medium mixture (2 and 10 min). The statistical significance of the obtained results was determined.     

Increased Sensitivity of the Microbiological Assay for Biotin by Lactobacillus plantarum

Addition of Tween 80 to biotin assay medium containing acid-hydrolyzed casein as the amino acid source caused marked growth of Lactobacillus plantarum ATCC 8014 in the absence of added biotin. This growth-promoting activity could be eliminated by treating the "vitamin-free" Casamino Acids (Difco) with activated charcoal (Darco G-60) at pH 3.5 for 30 to 60 min. Incorporation of Tween 80 and charcoal-purified Casamino Acids (PCA) into the assay medium (0.8 g and 27 g, respectively, per liter of single strength medium) in place of unpurified Casamino Acids resulted in a medium in which L. plantarum responded to 30 to 50 times less biotin over an extended linear response range (1.3 logs versus 1.0 log) than was required for similar growth in the standard medium. Endogenous growth in the modified medium was absent if the inoculum used was of low density, if it was prepared from biotin-deficient cells, and if the reagents used were free from contaminating traces of biotin. Assays of biological materials for biotin content using the standard medium and the Tween 80-PCA-modified medium resulted in nearly identical values for all samples tested.     

Autotrophic Growth of Thiobacillus acidophilus in the Presence of a Surface-Active Agent, Tween 80

Cellular protein, pH, dissolved oxygen concentration, and static surface tension were measured during growth of Thiobacillus acidophilus on elemental sulfur in the absence and presence of up to 5,000 mg of Tween 80 per liter. The decrease in pH and the increase in sulfate production were observed to be less accurate measurements of growth when compared with the increase in cellular protein. The doubling time of the bacterium decreased approximately 50% with the addition of 500 mg of Tween 80 per liter. The bacteria did not appear to synthesize any wetting agents as demonstrated by the constant surface tension of the medium during growth. Morphological alterations in the presence of Tween 80 were also observed.     

Growth Kinetics of Clostridium bifermentans and Its Ability to Degrade TNT Using an Inexpensive Alternative Medium

A strain of Clostridium bifermentans isolated from a munitions-supplemented anaerobic digester is known to degrade 2,4,6-trinitrotoluene (TNT) in rich media such as Brain Heart Infusion (BHI) broth. In order to make this biodegradation process commercially feasible, a new growth medium was developed. Corn steep liquor and molasses were selected as possible nitrogen and carbon sources. A medium containing 2.4% corn steep liquor and 0.4% molasses was chosen based on the value for maximum specific growth rate. The values of µ_m and K_s from continuous runs were of 0.029 min^-1 and 0.488 g/L, respectively. To reduce the overhead cost for maintaining an anoxic environment for this obligatory anaerobic bacterium, the threshold oxygen level under which the bacterium can survive was determined. The degradation of TNT was then carried out in a batch bioreactor, and in a continuously stirred tank bioreactor. The solubility of TNT was enhanced by using the surfactant, Tween 80, and the optimal concentration of Tween 80 was determined to be 2.5%. This concentration of Tween 80 increased TNT solubility by 100% while the effect on growth rate was minimal. Degradation of TNT was then carried out in a continuously stirred tank bioreactor and was found to follow Michaelis-Menten-type kinetics. Degradation of TNT was found to be possible under limited oxygen presence, but at a reduced degradation rate.     

Microthrix parvicella, a filamentous bacterium from activated sludge: growth on Tween 80 as carbon and energy source

Microthrix parvicella, cultivated in a medium with Tween 80 and Casamino acids, utilized only the oleic acid moiety of Tween 80 as carbon and energy source. The cell yield from Tween 80 was about 0.32 g dry weight of cells per g of Tween 80 consumed. As only the oleic acid moiety of Tween 80 was utilized, the cell yield from oleic acid was 1.3 g dry weight of cells per g oleic acid consumed. The amount of carbon produced as CO2 was less than 30% of the oleic acid-carbon and this low value was in agreement with the high cell yield. In batch culture M. parvicella stored large amounts of lipid material during the early growth phase. The fatty acids of the lipid globules were similar to the fatty acids supplied as carbon source. The percentage composition of the biomass changed to give C/N percentage ratios of about 15 during the early growth phase due to the high concentration of internal lipids and the low concentration of protein. The growth rate in batch culture was about 0.016 h-1 but was affected by the concentration of Casamino acids in the medium.     

Chemically defined medium for high yields of sterigmatocystin

Isolate of Aspergillus versicolor strain produced 138 g/ml of sterigmatocystin in a complete synthetic medium containing sucrose, salts, 1-phenylalanine, and Ca-pantothenate. The SSP (sucrose salts phenylalanine) medium apparently provided all necessary ingredients for the production of high levels of sterigmatocystin. For optimal sterigmatocystin formation, the amounts of sucrose and 1-phenylalanine were found to be 200 g and 5 g per liter, respectively. When Ca-pantothenate (0.01 g per liter) added, much higher amounts of sterigmatocystin were recovered, whereas CaCl₂ addition (0.01%) drastically reduced the yield. The high levels of sterigmatocystin were recovered in the cultures which incubated stationarily at 26 to 29 °C for over 12 days. Seven strains or isolates tested yielded high levels of sterigmatocystin in the SSP medium, whereas in each other media such as YES medium and rice medium only one isolate yielded highest amount of sterigmatocystin was found.     

Glucose and nutrient concentrations affect the expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes

Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K(2)HPO(4) reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.     

Medium optimization of a hydrophilic solvent‐stable protease from Serratia sp. SYBC H

Two statistical methods were used for medium optimization for a hydrophilic solvent‐stable protease production by Serratia sp. SYBC H with duckweed as the nitrogen source. Orthogonal design was applied to find the significant variables, then response surface methodology (RSM), including Box–Behnken central composite experiments, was used to determine the optimal concentrations and interaction of the significant variables. Results demonstrated that duckweed powder, wheat flour, Tween 80, sodium chloride had significant effects on the solvent‐stable protease production. The interaction between duckweed and wheat flour was significant. The optimal level of the variables for the maximum protease production was duckweed 43.9 g/L, wheat flour 20 g/L, sodium chloride 0.08 M, Tween 80 1% v/v, initial pH 11.0, and inoculum size 7% v/v. The maximum protease activity reached 1922.8 U/mL in the optimized medium, with about 18.3‐fold higher than that in the unoptimized medium. Most importantly, the protease from Serratia sp. SYBC H has successfully catalyzed the specific acylation of sucrose in a two‐solvent medium consisting of pyridine and n‐hexane (1:1, v/v), and non‐specific acylation of sucrose in anhydrous DMSO. These results demonstrated that the protease from Serratia sp. SYBC H is a solvent‐stable protease and it could be an ideal biocatalyst for sugar esters syntheses in non‐aqueous media.     

A SEAWATER MEDIUM FOR DINOFLAGELLATES AND THE NUTRITION OF CACHONINA NIEI1

Two media have been devised: an enriched seawater medium for culture of dinoflagellates and a defined medium for rapid growth of the dinoflagellate Cachonina niei. A wide range in salinity (10.23–42.38 g/liter NaCl) is tolerated by C. niei. Below 0.6 g/liter MgSO₄, 0.19 g/liter KCl, and 0.22 g/liter CaCl₂, the generation time greatly increases. Increase in MgSO₄ to 7.22 g/liter, KCl to 1.12 g/liter or CaCl₂ to 2.22 g/liter has little effect on generation time. The temperature optimum is 19–23 C. Saturating light intensity for growth is 1000 ft-c and for photosynthesis (determined manometrically) is slightly less than 2000 ft-c. Cachonina niei requires B₁₂ and thiamin. Neither silicate nor its competitive inhibitor germanate affects generation time or cell yield indicating silicon is not required. Of a variety of buffers tested, Tris is the best. Optimal growth occurs at pHs of 7.5–8.3. Glycerol is inhibitory and does not support dark growth.     

Carbon Concentration and Carbon-to-Nitrogen Ratio Influence Submerged-Culture Conidiation by the Potential Bioherbicide Colletotrichum truncatum NRRL 13737

We assessed the influence of various carbon concentrations and carbon-to-nitrogen (C:N) ratios on Colletotrichum truncatum NRRL 13737 conidium formation in submerged cultures grown in a basal salts medium containing various amounts of glucose and Casamino Acids. Under the nutritional conditions tested, the highest conidium concentrations were produced in media with carbon concentrations of 4.0 to 15.3 g/liter. High carbon concentrations (20.4 to 40.8 g/liter) inhibited sporulation and enhanced the formation of microsclerotiumlike hyphal masses. At all the carbon concentrations tested, a culture grown in a medium with a C:N ratio of 15:1 produced more conidia than cultures grown in media with C:N ratios of 40:1 or 5:1. While glucose exhaustion was often coincident with conidium formation, cultures containing residual glucose sporulated and those with high carbon concentrations (>25 g/liter) exhausted glucose without sporulation. Nitrogen source studies showed that the levels of C. truncatum NRRL 13737 conidiation were similar for all protein hydrolysates tested. Reduced conidiation occurred when amino acid and inorganic nitrogen sources were used. Of the nine carbon sources evaluated, acetate as the sole carbon source resulted in the lowest level of sporulation.     

Ethanol Inhibition Kinetics of Kluyveromyces marxianus Grown on Jerusalem Artichoke Juice

The kinetics of ethanol inhibition on cell growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 were studied during batch growth. The liquid medium contained 10% (wt/vol) inulin-type sugars derived from an extract of Jerusalem artichoke (Helianthus tuberosus) tubers, supplemented with small amounts of Tween 80, oleic acid, and corn steep liquor. Initial ethanol concentrations ranging from 0 to 80 g/liter in the liquid medium were used to study the inhibitory effect of ethanol on the following parameters: maximum specific growth rate (μ_max), cell and ethanol yields, and sugar utilization. It was found that as the initial ethanol concentration increased from 0 to 80 g/liter, and maximum specific growth rate of K. marxianus cells decreased from 0.42 to 0.09 h⁻¹, whereas the ethanol and cell yields and sugar utilization remained almost constant. A simple kinetic model was used to correlate the μ_max results and the rates of cell and ethanol production, and the appropriate constants were evaluated.     

Gentamicin-based medium for the isolation of group D streptococci and application of the medium to water analysis.

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH(2)PO(4), 2.0 g of NaHCO(2), 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO(3) (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

低温脂肪酶产生菌的筛选、产酶发酵及粗酶性质研究

利用含有Tween 80的琼脂平板和摇瓶发酵法,从若尔盖高原土壤中筛选产脂肪酶菌株.通过菌落形态和菌体特征观察初步对菌种进行鉴定,得到一株产低温脂肪酶的适冷菌Pseudomonassp.DL-B,并设计正交试验对该菌株的产酶发酵培养条件进行了优化.摇瓶实验表明,该菌株最适产酶发酵培养基为:蔗糖10 g/L,蛋白胨20 ...     

Incorporation of [(15)N] ammonia by the cellulolytic ruminal bacteria Fibrobacter succinogenes BL2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17

The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using (15)NH(3). At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of Fibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albus SY3 (46%) were derived from non-NH(3)-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH(3). More cell nitrogen was formed from NH(3) during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for F. succinogenes, and its (15)N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.     

Benefits from tween during enzymic hydrolysis of corn stover

Corn stover is a potential substrate for fermentation processes. Previous work with corn stover demonstrated that lime pretreatment rendered it digestible by cellulase; however, high sugar yields required very high enzyme loadings. Because cellulase is a significant cost in biomass conversion processes, the present study focused on improving the enzyme efficiency using Tween 20 and Tween 80; Tween 20 is slightly more effective than Tween 80. The recommended pretreatment conditions for the biomass remained unchanged regardless of whether Tween was added during the hydrolysis. The recommended Tween loading was 0.15 g Tween/g dry biomass. (The critical relationship was the Tween loading on the biomass, not the Tween concentration in solution.) The 72-h enzymic conversion of pretreated corn stover using 5 FPU cellulase/g dry biomass at 50 degrees C with Tween 20 as part of the medium was 0.85 g/g for cellulose, 0.66 g/g for xylan, and 0.75 for total polysaccharide; addition of Tween improved the cellulose, xylan, and total polysaccharide conversions by 42, 40, and 42%, respectively. Kinetic analyses showed that Tween improved the enzymic absorption constants, which increased the effective hydrolysis rate compared to hydrolysis without Tween. Furthermore, Tween prevented thermal deactivation of the enzymes, which allows for the kinetic advantage of higher temperature hydrolysis. Ultimate digestion studies showed higher conversions for samples containing Tween, indicating a substrate effect. It appears that Tween improves corn stover hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector. Copyright 1998 John Wiley & Sons, Inc.     

Improved co-oxidation of beta-carotene to beta-ionone using xanthine oxidase-generated reactive oxygen species in a multiphasic system

beta-Ionone, an aroma compound exhibiting flower notes, can be obtained from beta-carotene in a cooxidation system utilizing xanthine oxidase-generated reactive oxygen species (ROS). ROS have to be controlled as, although they can give rise to beta-ionone, they may also degrade it. In this work, the biotransformation of beta-carotene into beta-ionone was investigated in systems containing variable proportions of decane to extract beta-ionone before degradation. The use of 50% or 90% decane resulted in increased production yields. Tween 80, which was added to further improve the production, slightly decreased the reactivity of the medium and the extraction of beta-carotene, but increased the extraction of beta-ionone. In total, the addition of Tween 80 significantly improved the yields of conversion, which reached 34% with 50% decane and 2.5 g/L Tween 80 compared to 10% without decane and Tween 80. These results show that it is possible to control a ROS-mediated reaction by the addition of a solvent phase and by modifing the medium composition.     

Determination of ligninase activity in P. chrysosporium pellets with diffuse reflectance spectrophotometry

Diffuse reflectance spectrophotometry was applied for measuring ligninase activity in pellets of Phanerochaete chrysosporium. Enhanced ligninase activity in pellets and in growth medium were detected in cultures supplemented with oleic acid emulsified with Tween 80, Tween 80, hydrophilic (alcoholic) residue of Tween 80 hydrolysate, Tween 20, and (15OE)C_18:1. In cultures with low extracellular ligninase activity, low activity in pellets was also observed. Our results indicate that the stimulatory effect of the tested surfactants cannot be contributed solely to promotion of ligninases through the cell membrane. Correspondence to: D. Letan     

Culture conditions for the production of pectinolytic enzymes by the white-rot fungus Trametes trogii on a laboratory scale

Different cultural parameters that regulate pectinolytic enzyme production in vitro by Trametes trogii were studied. When grown in a medium containing pectin, T. trogii produced extracellular polymethylgalacturonase, polygalacturonase and pectin lyase but no pectate lyase activity. No significant differences in the maximum enzyme activities measured were observed with the addition of xylan, carboxymethylcellulose or both to the medium containing pectin. The addition of glucose to that medium considerably decreases all the activities studied, and in a medium with glucose as the sole carbon source no galacturonase activity could be measured, and pectin lyase activity was at its minimum. The low synthesis of pectin lyase in cultures containing glucose suggests that this enzyme is constitutive in contrast to the polygalacturonases that were not detected. The increase in pectin concentration stimulated growth and enzyme production. The highest specific activities were attained with the greatest concentration tested (15 g/l). Casamino acids were the best nitrogen source for enzyme production. Maximum growth was measured at pH 3.3; pH values of around 4.5 stimulated enzyme production, but high pectinase activities were also detected in media with more alkaline initial pH values (6.2 for galacturonases and 6.6 for lyases), probably owing to the specific induction of particular isoforms. In the range of 23 to 28°C, good results were obtained in growth as well as in enzyme production. The addition of Tween 80 promoted growth and gave the highest yield of polymethylgalacturonase and pectin lyase (0.37 and 36.2 E.U./ml, respectively). The highest polygalacturonase activity (1.1 E.U/ml) was achieved with polyethylene glycol. Tween 20 and Triton X-100 inhibited growth and pectinase production.     

Degradation of benzo[a]pyrene by the litter-decomposing basidiomycete Stropharia coronilla: role of manganese peroxidase

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn(2+)) supplied at a concentration of 200 micro M stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn(2+) than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn(2+). Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter(-1) was converted in an in vitro reaction solution containing 1 U of MnP ml(-1) within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter(-1)) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).