Accurate and rapid viability assessment of Trichoderma harzianum using fluorescence-based digital image analysis

Fluorescence microscopy and image analysis were evaluated in order to assess the viability of Trichoderma harzianum, an economically important filamentous fungus. After the evaluation of the two most commonly used fluorochromes, acridine orange (AO) and fluorescein diacetate (FDA) as metabolic indicator stains, AO gave ambiguous results and therefore FDA was chosen. The lower stability at room temperature and fast fluorescence intensity decay (50% after only 30 s of illumination in UV light) could be overcome by the use of a digital image acquisition system including frame grabber and a video camera. Fresh (live) fungal hyphae emitted bright green fluorescence when stained with this dye (7.5 microg/L), whereas a total absence of fluorescence was observed when using sterilized (dead) fungal cells. Fresh cells were subjected to different lethal and sublethal treatments and the percentage of FDA stained fluorescent hyphae was then measured over the total hyphal area (% of FDA-stained area) by image analysis. At the same time, samples were cultivated in shake flasks in order to correlate this % of FDA-stained area with its growth rate, a functional indicator of viability. The linear correlation (r = 0.979) was: growth rate (g/L x h) = 2.25 x 10(-3) (% of FDA-stained area). This method was used to evaluate the viability of the fungus under two different fermentation conditions in a 10-L bioreactor. Estimated viable biomass during fermentation was strongly influenced by the process conditions. The use of FDA, with computer-aided quantitative image analysis, has made it possible to rapidly and reliably quantify the viability of T. harzianum.     

Phosphatase activity ofPenicillium citrinum submerged batch cultures and its relationship to fungal activity

Acid and alkaline phosphatase activities were evaluated using batch fermenter cultues ofPenicillium citrinum, an organism used in studies of fungal functioning in soil. Fungal activity was assessed by monitoring rates of O₂ utilization, glucose utilization, dry weight changes over time, and lengths of FDA-stained hyphae. At low growth rates (7 g dry wt increases·h^–1·ml^–1) and low culture activity, phosphatase activity at both pH 8.5 and 5.5 tended to decrease with culture age, with the exception that phosphatase activity at pH 8.5 peaked during early stationary phase. At higher growth rates (25 g dry wt increase·h^–1·ml^–1) and high culture activity, phosphatase activity tended to remain constant throughout the course of the experiment. The relationship between phosphatase activity and other measures of fungal activity was consistent only at low growth rates for acid phosphatase. These results suggest that phosphatase measurements will be of limited utility in assessing activity, except at low growth rates.     

Circulating lupus-type anticoagulant, a risk factor for thrombosis by inhibition of protein C activation

The lupus anticoagulant is a risk factor of thrombosis. The non thrombogenic endothelial surface could be a target for the lupus anticoagulant. We have investigated the effect of purified immunoglobulins G of five patients with LA on the thrombomodulin activity of cultured human endothelial cells from umbilical cord vein. The rate of activation of purified protein C (PC) (30 nM) by the endothelial cells in the presence of thrombin (0.1 U/dish) has been measured by hydrolysis of substrate S 2366. Activated PC has been 7.37 +/- 0.78 pmoles X ml-1 X h-1 in the presence of buffer and 7.2 +/- 0.78 pmoles X ml-1 X h-1 in the presence of control IgG (2 mg/dish). Heat aggregated IgG did not induce any significant change. Patient's IgG lowered significantly the rate of PC activation (4.86 +/- 1.04 pmoles X ml-1 X h-1, p less than 0.001). Fab fragment from two of these patient's IgG displayed the same inhibition. Moreover neutralization of this effect was obtained by addition of phospholipids (70% phosphatidylcholine, 30% phosphatidylserine) in excess to patient's IgG. Activation of PC has been also performed using purified rabbit thrombomodulin and a similar inhibition by patient's IgG was found. These results seem to indicate that antibodies present in the IgG fractions containing LA could be directed against phospholipids associated to thrombomodulin activity. Reduction of PC activation if present in the patients with LA could play a role in the occurrence of thrombosis.     

Monoclonal antibodies incorporated into Neotyphodium coenophialum fungal cultures: inhibition of fungal growth and stability of antibodies.

Three monoclonal antibodies (mAbs) produced against proteins from the tall fescue (Festuca arundinacea Schreb.) fungal endophyte Neotyphodium coenophialum hybridize exclusively to a fungal protein under denaturing conditions. The protein is approximately 88 kDa in size. These mAbs were individually incorporated into liquid medium to determine their effects on fungal growth in culture. Neotyphodium-specific mAbs inhibited fungal growth for the duration of the study. Fungal cultures grown in the presence of Neotyphodium-naive mAbs or in the absence of all mAbs grew unimpeded. Bright-field microscopy and immunohistochemical studies of cultures containing Neotyphodium-specific mAbs revealed a change in mycelia morphology with clumps exhibiting a gelatinous matrix containing sparse hyphae, while cultures receiving Neotyphodium-naive mAbs in medium demonstrated unrestricted growth with overlapping and branched hyphae. In liquid culture devoid of fungal isolates, mAbs were stable and detected throughout the experiment, but were below threshold detection levels within 15 min following inclusion in liquid cultures containing Neotyphodium spp., indicating rapid binding to fungal mycelia. Monoclonal antibodies may provide a new method to help control plant pathogenic fungi where chemical or genetic means are not feasible.     

Effect of carbon dioxide on growth of Pseudomonas putida ATCC 11172 on asparagine, citrate, glucose, and lactate in batch and continuous culture

The growth of Pseudomonas putida ATCC 11172 on L-asparagine, citrate, D-glucose, and L-lactate was followed in air and in 40% CO2 + air, using batch and carbon-limited continuous cultures. Batch cultures in air utilized a mixture of the carbon sources simultaneously. However, a change to 40% CO2 favoured the utilization of glucose. The maximum specific growth rate (mumax) in air was about 0.3 h-1 on glucose and 0.6 h-1 on the other carbon sources. In CO2, the mumax for glucose was reduced by 16% compared with almost 60-70% for the others. An order of preference for the different carbon sources in continuous cultures was determined by comparing the dilution rates at which the different carbon sources started to appear in the effluent. Glucose was the first compound to appear as the dilution rate increased (lowest preference when grown in air). In 40% CO2, the mumax for glucose was slightly higher than the others and the recorded preference for glucose in continuous culture was equal to that for citrate but was somewhat lower than that of lactate and asparagine. D-Gluconate and glucono-delta-lactone were produced as a step in the utilization of glucose. The D-gluconate production was enhanced by CO2.     

Succinate dehydrogenase activity of external and internal hyphae of a vesicular-arbuscular mycorrhizal fungus,Glomus mosseae (Nicol. & Gerd.) Gerdmann and Trappe,during mycorrhizal colonization of roots of leek (Allium porrum L.), as revealed by in situ histochemical staining

The succinate dehydrogenase (SDH) activity of hyphae of the vesicular-arbuscular (VA) mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerdmann and Trappe, in symbiotic association with leek (Allium porrum L.) roots, was investigated by histochemical staining in situ. Leek seedlings were transplanted to sand culture and inoculated with spores of G. mosseae placed just below the base of the stem. At intervals (14, 25, 35 and 60 days) after transplanting, the growth medium of seedlings was flooded with nitro blue tetrazolium chloride solution, thereby displacing the nutrient solution. This allowed sites of SDH activity of external and internal fungal structures of the mycorrhizas to be stained without physically disturbing the symbiotic system. After counterstaining harvested roots and mycelium with acid fuchsin, it was possible to differentiate clearly metabolically active and inactive regions of the fungus. The lengths of external hyphae and infected root both increased nearly exponentially, and were in constant proportion (1.4 m hyphae per cm of infected root) for up to 60 days. The percentage length of external hyphae with SDH activity remained almost constant (80%). In each infected length of root there was a gradation of SDH activity from inactive distal (older) hyphae to uniformly active proximal (younger) hyphae. These findings are discussed in relation to the symbiotic activity of the mycobiont.Deceased     

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.     

The effects of temperature, pH and growth rate on secondary metabolism in Streptomyces thermoviolaceus grown in a chemostat.

Streptomyces thermoviolaceus was grown in a chemostat under conditions of glutamate limitation. The effects of growth rate on production of the antibiotic granaticin, extracellular protein and protease activity as components of secondary metabolism were studied at 37, 45 and 50 degrees C. The amount of each secondary metabolite synthesized was highly dependent on growth rate and temperature. Granaticin yields were highest at growth rates of 0.1 to 0.15 h-1 at 37 degrees C, 0.175 h-1 at 45 degrees C and 0.045 h-1 at 50 degrees C. Protease activity of culture supernatants responded to low nutrient concentration and/or low growth rate. Measurements of extracellular protein revealed complex changes in amount which were dependent on growth rate and temperature. At 45 degrees C and a growth rate of 0.15 h-1, biomass yield was highest between pH 5.5 to 6.5 whereas granaticin synthesis was low at pH 5.5 and rose to highest values at between pH 6.5 and 7.5.     

The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses

The development of media free of serum and animal or human proteins is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. In order to reduce the risk of contamination, we have modified the serum free medium MDSS2, a very efficient serum free medium for the production of various biologicals including experimental vaccines using different cell lines (Merten et al., 1994), by replacing the animal derived products by plant extracts. The new serum and animal protein free medium (MDSS2N) can be efficiently used for biomass production of various cell lines. These cells grow equally well or better in this new serum-free medium than in the old formulation (MDSS2): • BHK-21/BRS cells, adapted to MDSS2N, showed an overall specific growth rate of 0.0197 h-1 (μ_max = 0.0510±0.0058 h-1), whereas those cultivated in MDSS2 grew with an average specific growth rate of 0.0179 h-1 (μ_max = 0.0305±0.0177 h-1). • Vero cells grew with an average specific growth rate of 0.0159 h-1 and 0.0153 h-1 in MDSS2 and MDSS2N, respectively. Very similar growth rates were obtained in microcarrier cultures in stirred tank reactors: the specific growth rates were 0.0161 h-1 and 0.0166 h-1 for MDSS2 and MDSS2N cultures, respectively. • For MDCK cells, when cultured on microcarriers in bioreactors, a higher average specific growth rate was observed in MDSS2N than in MDSS2; values of 0.0248 h-1 and 0.0168 h-1, respectively, were obtained. The capacity of MDSS2N to support the production of different viruses was equally evaluated and it could be established that for certain viruses there are no or insignificant differences between MDSS2N and MDSS2 (influenza and polio virus), whereas, the production of rabies virus is somewhat reduced in MDSS2N when compared to MDSS2. The use of MDSS2N for cell culture and the production of various viruses is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Improvement of Y-values of Hansenula polymorpha growth on methanol by simultaneous utilization of glucose

The simultaneous utilization of methanol and glucose by Hansenula polymorpha MH20 was investigated in chemostat (C-limited) cultivation. The mixed-substrate utilization results in biomass yields which are greater up to 20 to 25% as expected assuming an additive growth on both substrates. This is referred to as an auxiliary-substrate effect. Additionally, methanol can be utilized at higher growth rates in the presence of glucose compared to those obtained on this substrate alone. The extend of the auxiliary-substrate effect and the optimum ratio of substrates to reach this effect depend on dilution rate. The greatest stimulation in yield is obtained at D approximately 0.1 h-1, after raising the dilution rate this effect diminishes. At a rate of 0.1 h-1 the optimum mixed-substrate ratio of methanol: glucose is 7:1 (g). By increasing the growth rate the ratio changes toward glucose and reached a value of 1:1 (g) at D = 0.3 h-1. This change in the optimum ratio correlates with diminution in yield coefficient of methanol accompanying an increase in growth rate greater than 0.15 h-1. Energy balances of the utilization of the single substrates are used for interpretation of these results. From this it is evident that methanol does not play the role of an energy-rich substrate in the metabolism of yeast. Rather glucose is the energy-providing substrate in this combination.     

Effect of growth rate and cell shape on the peptidoglycan composition in Escherichia coli.

The muropeptide composition of peptidoglycan from Escherichia coli W7 cultivated at different growth rates in chemostat cultures was compared by using high-pressure liquid chromatography. At a low growth rate (D = 0.1 h-1), about 40% more covalently bound lipoprotein and at least twofold more diaminopimelyl-diaminopimelic acid cross-bridges were found than at a high growth rate (D = 0.8 h-1). The total degree of cross-linkage was only slightly increased, and the fraction of trimeric muropeptides and the average length of the glycan chains were not changed significantly. Analysis of the peptidoglycan from a morphological variant strain of W7 revealed that the altered peptidoglycan composition in slowly growing W7 cells was not correlated with the observation that these cells, due to their decreased cell length, were relatively enriched in polar material. In fact, our results suggested that peptidoglycan forming cell poles is chemically identical to that forming lateral wall.     

Physiology of the growth and supersynthesis of DNA-polymerase in Escherichia coli during 2-stage continuous cultivation

The physiology of growth under the conditions of batch and continuous cultivation was studied with the recombinant strain of Escherichia coli CM 5199 capable of DNA polymerase I superproduction. The specific growth rate of the strain is 0.8 h-1 under the conditions of continuous cultivation which is almost 2.5 times greater than that in the exponential phase of batch cultivation. When the strain was cultivated at a flow rate above 0.3 h-1, the biomass concentration in the fermenter decreased and the culture was no more limited by the carbon source in the absence of other growth limiting components of the medium. Apparently, the metabolic product ceased to inhibit high growth rates of the culture under the conditions of continuous cultivation. The rate of DNA polymerase synthesis correlated with the specific growth rate and the respiration activity of the culture when the lambda pol A prophage was induced in the cells. The authors discuss the effectiveness of ribosome operation in the cells at a growth rate of 0.05 to 0.3 h-1 and the content of ribosomes at a higher growth rate in relation to DNA polymerase I synthesis.     

贵州栽培笃斯越橘DSE真菌、AM真菌与ERM真菌空间分布研究

为探明贵州省栽培笃斯越橘根系深色有隔内生真菌（dark septate endophytes,DSE）、丛枝菌根（arbuscular mycorrhiza,AM）真菌与欧石南菌根（ericoid mycorrhiza,ERM）真菌的定殖及地理分布情况,揭示共生真菌在栽培笃斯越橘生长中的地位,本研究在贵州省笃斯越橘主栽区麻江县、凤岗县和高坡乡分别选取主栽品种圆蓝、粉蓝、奥尼尔和莱格西的根样及根围土样,观测不同地区不同品种根样DSE真菌、AM真菌和ERM真菌的定殖结构和定殖率,并测定土样土壤理化性质,分析不同真菌与土壤因子相关性。结果表明,3个地区的4个笃斯越橘品种均有DSE真菌、AM真菌和ERM真菌定殖,栽培笃斯越橘能与3类真菌形成共生关系,平均定殖率分别为61.11%、25.55%和22.50%。DSE真菌定殖率：高坡（62.50%）>麻江（61.66%）>凤岗（59.16%）;AM真菌定殖率：凤岗（34.14%）>麻江（25.83%）>高坡（16.66%）;ERM真菌定殖率：高坡（35.00%）>凤岗（20.00%）>麻江（12.5%）。相关性分析表明,DSE真菌中的菌丝与微菌核的定殖率呈负相关,AM真菌的总定殖率及定殖强度与微菌核的定殖率呈极显著正相关,与DSE真菌菌丝的定殖率呈负相关。ERM真菌总定殖率与DSE真菌菌丝的定殖率及AM真菌定殖强度呈极显著正相关,与DSE真菌总定殖率呈显著正相关。土壤有效磷与DSE真菌和ERM真菌总定殖率呈显著正相关,与AM真菌定殖率呈显著负相关。土壤铵态氮与DSE真菌中微菌核结构定殖率及AM真菌定殖率呈极显著正相关,与ERM真菌定殖率呈极显著负相关。土壤pH值与DSE定殖强度呈显著负相关,与ERM定殖强度呈极显著正相关。本研究分析比较贵州省3个笃斯越橘种植基地不同品种栽培笃斯越橘DSE真菌、AM真菌和ERM真菌的定殖及其与土壤理化性质之间的相关性,为贵州省栽培笃斯越橘的管理和发展提供技术基础和理论依据。     

Influence of growth rate and iron limitation on the expression of outer membrane proteins and enterobactin by Klebsiella pneumoniae grown in continuous culture.

The influence of the growth rate on outer membrane protein composition and enterobactin production was studied with Klebsiella pneumoniae grown under conditions of iron limitation in chemostats. More enterobactin was produced at fast (D = 0.4 h-1) and slow (D = 0.1 h-1) growth rates in continuous cultures than in either logarithmic- or stationary-phase batch cultures. When the growth rate was controlled under conditions of carbon limitation and the iron level was reduced to 0.5 microM, the iron-regulated outer membrane proteins and enterobactin were induced at the fast growth rate. At the slow growth rate, although the iron-regulated outer membrane proteins were barely visible, a significant level of enterobactin was still produced. These results suggest that under conditions of either carbon or iron limitation, the growth rate can influence the induction of the high-affinity iron uptake system of K. pneumoniae. Other outer membrane proteins, including a 39-kilodalton peptidoglycan-associated protein, were found to vary with the growth rate and nutrient limitation.     

Inducible and constitutive formation of fructanase in batch and continuous cultures of Streptococcus mutans

The production of extracellular beta-D-fructanase by several strains of Streptococcus mutans was studied in continuous culture. When glucose was the limiting nutrient, S. mutans K1-R and OMZ176 accumulated fructanase to maximum levels at low growth rates (dilution rate 0.05-0.10 h-1), due to the longer residence times of the bacteria in the culture vessel under these conditions. Extracellular fructanase activity was greater than has been previously reported for batch cultures. The rate of fructanase production for both S. mutans strains K1-R and OMZ176 increased with increasing growth rate when glucose was limiting. Under conditions of glucose sufficiency, the rate of fructanase production was always lower than in cultures where glucose was limiting, irrespective of the growth rate. Cultures of S. mutans Ingbritt (serotype c) grown with sorbitol- or glucose-limitation synthesized fructanase at a very low basal rate. When fructose was the limiting carbohydrate the enzyme was induced with a maximum rate of production occurring at a dilution rate of 0.40 h-1. Strains of S. mutans from other serotypes (a, d, d/g) were either not affected by changing the limiting sugar from glucose to fructose or else fructanase activity was slightly decreased in the fructose-limited medium. Fructanases from various strains of S. mutans readily hydrolysed (2----6)-beta-D-fructans, but all possessed the ability to hydrolyse (2----1)-beta-D-fructans to varying degrees.     

Maize ribosome-inactivating protein inhibits normal development of Aspergillus nidulans and Aspergillus flavus

The abundant maize kernel ribosome-inactivating protein 1 (RIP1) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A microculture assay was developed to monitor fungal growth and development after treatment of conidia with RIP1 or control proteins. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well-characterized nonpathogenic species, were treated with RIP1 protein. Treatment with a RIP1 mutant protein that lacked enzymatic ribosome-inactivating activity caused no observable effects. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed only one growing hyphal tip per conidium. These results indicate that both fungi were affected by RIP1 treatment, but the lysis seen with treatment of A. nidulans was apparently avoided by A. flavus. A developmental time course revealed that both fungal species were affected by RIP1 at the postdivisional growth stage. The inhibitory activity of RIP1 against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion.     

Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis

Cellulase production was examined in two strains of Neocallimastix frontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50 degrees C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-1 (1 U represents 1 microgram of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-1. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 X 10(3) to 10.4 X 10(4) U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-1. The range for PN-2 was 8.1 X 10(3) to 11 X 10(4) U g-1. Shaking cultures improved yields for strain PN-1 but not for PN-2. Decreased CMCase production at high initial cellulose concentrations concurred with accumulation of glucose, and addition of glucose (4 mg ml-1) to cultures grown on low cellulose in which none of the sugar accumulated repressed CMCase. Adsorption of CMCase was excluded as a likely explanation for decreased yields at high initial cellulose as only a low proportion (less than 20%) of the enzyme was adsorbed onto the growth substrate. Exoglucanase, measured with alkali-treated Sigmacell or Avicel, gave low levels of activity in the culture fluid (less than 2 U ml-1) and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity.(ABSTRACT TRUNCATED AT 250 WORDS)