Extracellular biosynthesis of silver nanoparticles using a novel and non-pathogenic fungus,Neurospora intermedia: controlled synthesis and antibacterial activity

In the present study, the biosynthesis of silver nanoparticles (AgNPs) using Neurospora intermedia, as a new non-pathogenic fungus was investigated. For determination of biomass harvesting time, the effect of fungal incubation period on nanoparticle formation was investigated using UV–visible spectroscopy. Then, AgNPs were synthesized using both culture supernatant and cell-free filtrate of the fungus. Two different volume ratios (1:100 and 1:1) of the culture supernatant to the silver nitrate were employed for AgNP synthesis. In addition, cell-free filtrate and silver nitrate were mixed in presence and absence of light. Smallest average size and highest productivity were obtained when using equal volumes of the culture supernatant and silver nitrate solution as confirmed by UV–visible spectra of colloidal AgNPs. Comparing the UV–visible spectra revealed that using cell-free filtrate for AgNP synthesis resulted in the formation of particles with higher stability and monodispersity than using culture supernatant. The absence of light in cell-free filtrate mediated synthesis led to the formation of nanoparticles with the lowest rate and the highest monodispersity. The presence of elemental silver in all prepared samples was confirmed using EDX, while the crystalline nature of synthesized particles was verified by XRD. FTIR results showed the presence of functional groups which reduce Ag⁺ and stabilize AgNPs. The presence of nitrate reductase was confirmed in the cell-free filtrate of the fungus suggesting the potential role of this enzyme in AgNP synthesis. Synthesized particles showed significant antibacterial activity against E. coli as confirmed by examining the growth curve of bacterial cells exposed to AgNPs.     

The peculiarities of succinic acid production from rapeseed oil by Yarrowia lipolytica yeast

The process of succinic acid (SA) production represents the combination of microbial synthesis of α-ketoglutaric acid from rapeseed oil by yeast Yarrowia lipolytica VKM Y-2412 and subsequent decarboxylation of α-ketoglutaric acid by hydrogen peroxide to SA that leads to the production of 69.0 g l^?1 of SA and 1.36 g l^?1 of acetic acid. SA was isolated from the culture broth filtrate in a crystalline form. The SA recovery from the culture filtrate has certain difficulties due to the presence of residual triglycerides of rapeseed oil. The effect of different methods of the culture filtrate treatment and various sorption materials on the coagulation of triglycerides was studied, and as a result, the precipitation of residual triglycerides by acetone was chosen. The subsequent isolation procedures involved the decomposition of H₂O₂ in the filtrate followed by filtrate bleaching and acidification with a mineral acid, evaporation of filtrate, and SA extraction with ethanol from the residue. The purity of crystalline SA isolated from the culture broth filtrate achieved 97.6–100 %. The product yield varied from 62.6 to 71.6 % depending on the acidity of the supernatant.     

Effect of Streptococcus parvulus and Peptostreptococcus magnus on cytotoxin levels of Clostridium difficile in anaerobic continuous flow culture

An anaerobic continuous flow (CF) culture method was used in order to study the effect of Peptostreptococcus magnus and Streptococcus parvulus, anaerobic gram-positive cocci which are members of intestinal bacterial flora, on growth and cytotoxin-activity of Clostridium difficile. The growth- and the cytotoxin activity-patterns of C. difficile in an established CF culture of P. magnus were similar to those of C. difficile alone. On the other hand, in the mixed culture system of C. difficile and S. parvulus, the cytotoxin levels were significantly lower as compared with C. difficile alone in spite of the fact that no differences existed between growth of C. difficile in mixed and single culture systems. The culture filtrate of P. magnus did not influence the growth and cytotoxin production of C. difficile, nor did that of S. parvulus have any effect on growth of C. difficile in static culture. The cytotoxin activity of C. difficile was, however, suppressed by the culture filtrate of S. parvulus. Furthermore, when P. magnus or S. parvulus was statically cultured in a medium containing cytotoxic culture filtrate of C. difficile, the toxin in the medium was not inactivated.     

Kinetic modeling of kefiran production in mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae

Growth and kefiran production rates of Lactobacillus kefiranofaciens were significantly enhanced in a mixed culture with Saccharomyces cerevisiae as compared with those in a pure culture. Because a positive effect on growth and kefiran production of L. kefiranofaciens in a mixed culture was observed, the elucidation of interaction between L. kefiranofaciens and S. cerevisiae may lead to higher productivity. Hence, microbial performance of each strain was investigated and analyzed by a mathematical model. The mathematical model for kefiran fermentation in a mixed culture of L. kefiranofaciens and S. cerevisiae was established, and the impact of S. cerevisiae on cell growth, kefiran formation, and substrate assimilation of L. kefiranofaciens were considered. The behavior of L. kefiranofaciens in a mixed culture was predicted using a developed mathematical model in this work, and the predictions were compared with the results from mixed culture experiments. The overall mathematical model is capable of describing the behavior of S. cerevisiae in a mixed culture as a lactic acid consumer, nitrogen source competitor and protective function inducer for L. kefiranofaciens. Furthermore, the constructed model described the phenomena in mixed cultures under aerobic and anaerobic conditions. Finally, the optimal inoculation ratios of S. cerevisiae to L. kefiranofaciens at 7-fold and 10-fold under aerobic and anaerobic conditions were obtained by applying the mixed culture model, respectively.     

Immunoelectrophoresis of mycobacterial antigens

Polyvalent antiserum to culture filtrate of H37 Ra M. tuberculosis was raised in rabbits. Monospecific antiserum was raised against M. tuberculosis antigen-5, prepared from the culture filtrates by immunoabsorbent affinity chromatography. On immunoelectrophoresis, antigen-5 demonstrated single precipitin arc against polyvalent and monospecific antisera. The culture filtrate antigen demonstrated multiple precipitin arcs against polyvalent antiserum and single precipitin are against monospecific antiserum. Antigen-5 could be isolated and characterized from the culture filtrate of H37 Ra M. tuberculosis. Immunoelectrophoresis could be one of the method to characterize the mycobacterial antigens prepared in the laboratory.     

Isolation of Mycobacterium tuberculosis protein antigens ES-3 1, ES-43 and EST-6 of diagnostic interest from tubercle bacilli by affinity chromatography

Immunodiagnostically useful M. tuberculosis H37Ra protein antigens ES-31, ES-43 and EST-6 were isolated from detergent soluble sonicate (DSS) antigen using monospecific antibodies by affinity chromatography and compared with similar antigens isolated from M. tuberculosis culture filtrate for seroreactivity in tuberculosis sera by Indirect Enzyme Linked Immunosorbent Assay. Recovery of affinity purified ES-31, ES-43 and EST-6 antigen from DSS antigen was approximately 3, 3.5 and 4% respectively, compared to 10, 9 and 6.3% from culture filtrate. Affinity purified ES-31, ES-43 and EST-6 antigens from both culture filtrate as well as DSS antigen showed similar seroreactivity with overall sensitivity 85, 80 and 75% respectively and specificity of 85% at optimum concentration of 50 pg protein of each antigen. The results suggest that DSS antigen may be a promising antigen source for isolating antigens of diagnostic interest obviating the need for cumbersome, time-consuming culture techniques of mycobacteria.     

Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.     

Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi

Indigenous actinomycetes isolated from rhizosphere soils were assessed for in vitro antagonism against Colletotrichum gloeosporioides and Sclerotium rolfsii. A potent antagonist against both plant pathogenic fungi, designated SRA14, was selected and identified as Streptomyces hygroscopicus. The strain SRA14 highly produced extracellular chitinase and β-1,3-glucanase during the exponential and late exponential phases, respectively. Culture filtrates collected from the exponential and stationary phases inhibited the growth of both the fungi tested, indicating that growth suppression was due to extracellular antifungal metabolites present in culture filtrates. The percentage of growth inhibition by the stationary culture filtrate was significantly higher than that of exponential culture filtrate. Morphological changes such as hyphal swelling and abnormal shapes were observed in fungi grown on potato dextrose agar that contained the culture filtrates. However, the antifungal activity of exponential culture filtrates against both the experimental fungi was significantly reduced after boiling or treatment with proteinase K. There was no significant decrease in the percentage of fungal growth inhibition by the stationary culture filtrate that was treated as above. These data indicated that the antifungal potential of the exponential culture filtrate was mainly due to the presence of extracellular chitinase enzyme, whereas the antifungal activity of the stationary culture filtrate involved the action of unknown thermostable antifungal compound(s).     

Protective and other biological properties of Bacillus anthracis soluble antigens

The soluble antigens were explored of the culture filtrate (CF) derived during static growth of B. anthracis vaccine strain 34F2 on a medium containing casein hydrolysate. Electrofocusing of CF preparations revealed that the protective activity was distributed over a wide range of pH 3-7. The most pronounced and stable protective activity was observed at pH 4.6-4.8. Following toxin factors were isolated and identified: protective antigen (87 kD), oedema factor (87 kD) and lethal factor (78-81 kD). The greatest protective activity was associated with antigens characterized by a molecular weight of 78-87 kD and toxic activity. Preparations of the oedema and lethal factors had the same protective activity as protective antigen (PA) preparations. Other CF soluble antigens protected about 30% of immunized guinea pigs. A protein was isolated with a molecular weight of 80 kD and isoelectric point at pH 5.3-5.7 which was not toxic and did not form toxic mixtures in association with other toxin factors; this protein featured a high immunogenic activity, however, it protected only 31% of immunized animals. Factors are analyzed which determine differences in the protective effects of live and chemical vaccines.     

Enhanced α-ketoglutaric acid production and recovery in Yarrowia lipolytica yeast by effective pH controlling

The replacement of chemical synthesis by environmentally friendly energy-efficient technologies for production of valuable metabolites is a principal strategy of developing biotechnological industry all over the world. In the present study, we develop a method for α-ketoglutaric acid (KGA) production from rapeseed oil with the use of Yarrowia lipolytica yeast. Sixty strains of Y. lipolytica yeasts were tested for their ability to produce KGA, and the strain Y. lipolytica 212 (Y. lipolytica VKM Y-2412) was selected as a promising KGA producer. Using a three-stage pH controlling, in which pH was 4.5 in the growth phase, then since 72 to 144 h, pH was maintained at 3.5 and in the later phase of acid production, the titration by KOH was switch off, selected strain produced 106.5 g l^?1 of KGA with mass yield of 0.95 g g^?1. KGA in the form of monopotassium salt was isolated from the culture broth and purified. The isolation procedure involved separation of biomass, extraction of residual triglycerides, filtrate bleaching, and acidification with mineral acid (to pH 2.8–3.4), concentration, precipitation of mineral salts, and crystallization of the product. The purity of KGA isolated from the culture filtrate reached 99.1 %.     

Ethylene production from l-methionine by Cryptococcus albidus

Cryptococcus albidus IFO 0939 was selected from microorganisms producing ethylene from l-methionine in a culture medium. When methionine was excluded from the culture medium of C. albidus, there was little production of ethylene. Ethylene production in a methionine-containing culture medium occurred for a brief period at the end of the growth phase. 2-oxo-4-methylthiobutyric acid (KMBA), a deaminated product of methionine, accumulated in the culture filtrate. An ethylene-forming enzyme was partially purified from C. albidus by means of DEAE-Sepharose CL-6B ion exchange chromatography, and a cell-free ethylene-forming system was constructed. Using this system, the precursor of ethylene was found to be KMBA and essential factors were NAD(P)H, Fe³⁺, EDTA and oxygen.     

Evaluation of in vitro free radical scavenging potential of Streptomyces sp. AM-S1 culture filtrate

The present study was carried out to determine the free radical scavenging potential of culture filtrate of Streptomyces sp. AM-S1. Antioxidant activity of culture filtrate, lyophilized culture filtrate and ethyl acetate extract of Streptomyces sp. AM-S1 was determined by various in vitro assays such as ferric reducing power assay, phosphomolybdenum reduction, DPPH and ABTS radical scavenging activities. The results revealed that the culture filtrate of Streptomyces sp. AM-S1 effectively scavenged DPPH (IC₅₀ 90.2 μl/ml) and ABTS (IC₅₀ 13.2 μl/ml) radicals in a concentration dependent manner. In all the assays, ethyl acetate extract registered higher antioxidant activity when compared with the lyophilized culture filtrate (LCF). In addition, ethyl acetate extract (1123.4 μmole Fe(II)/mg extract) exhibited higher ferric reducing activity than the standard BHA (814.4 μmole Fe(II)/mg extract). Further works are needed on the isolation and identification of antioxidant molecules from the ethyl acetate extract of Streptomyces sp. AM-S1 culture filtrate.     

Studies on the oxidation of tannins byAspergillus flavus

A method for the estimation of tannin in presence of catechin, pyrogallol, protocatechuic acid and gallic acid using polyamide column chromatography was developed. Tannin added to the growing culture ofAspergillus flavus was oxidised to different extents depending on the duration of incubation. The oxidised compound was identified in the culture filtrate as a polymerised product of tannin.     

Immune responses and protective efficacy induced by 85B antigen and early secreted antigenic target-6 kDa antigen fusion protein secreted by recombinant bacille Calmette-Guérin

In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin (rBCG) strains expressing an 85B antigen (Ag85B) and early secreted antigenic target-6 kDa antigen (ESAT6) of Mycobacterium tuberculosis (MTB) fusion protein. Both rBCG strains have the same protein insertion but in a different order (Ag85B-ESAT6 and ESAT6-Ag85B). The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B-ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The gamma-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group (p<0.05). Four weeks after vaccination, mice were infected with M. tuberculosis H37Rv and a dramatic reduction in the numbers of MTB colony forming units in the spleens and lungs was observed in the two rBCG immunization groups. Although these rBCG strains were more immunogenic, their protective effect was comparable to the classical BCG strain, and there were no significant differences between two rBCG groups (p>0.05).     

Cytokinin Production by Bradyrhizobium japonicum

Although there is considerable circumstantial evidence for the involvement of cytokinins in legume nodulation, the cytokinins produced by rhizobia have not been well characterized. Bradyrhizobium japonicum 61A68, a bacterium which nodulates soybean (Glycine max [L.] Merr.), was grown in defined medium. Cytokinins were purified from the culture medium by Amberlite XAD-2 chromatography and fractionated by column chromatography on Sephadex LH-20 in 35% ethanol. Pooled fractions from the Sephadex column were analyzed for cytokinin activity with the tobacco callus bioassay. Cytokinin activity was observed in fractions corresponding to the elution volumes of zeatin, ribosylzeatin, and methylthiozeatin. No activity corresponding to the elution volumes of isopentenyladenine or its riboside was found. Total cytokinin activity in the B. japonicum culture filtrate was equivalent to approximately 1 microgram of kinetin per liter. Transfer RNA was isolated from B. japonicum cells by phenol extraction, followed by potassium acetate extraction, cetyltrimethylammonium bromide precipitation, and DEAE cellulose chromatography. Transfer RNA was enzymically hydrolyzed to nucleosides. High performance liquid chromatographic analysis of cytokinin nucleosides showed peaks corresponding to the retention times of trans-ribosylzeatin, methylthioribosylzeatin, isopentenyladenosine, and methylthioisopentenyladenosine. Analysis of the tRNA hydrolysate by Sephadex LH-20 chromatography and tobacco bioassay showed cytokinin activity in fractions corresponding to ribosylzeatin, methylthioribosylzeatin, and isopentenyladenosine. The presence of the trans isomer of ribosylzeatin was also determined by enzyme immunoassay.     

Characteristics of insecticidal substances from the entomopathogenic fungus Metarhizium pinghaense 15R against cotton aphid in Korea

Aphids (Hemiptera: Aphididae) are agricultural pests for a wide range of economically important crops worldwide. Entomopathogenic fungi have been studied and developed as a biological agents to control hard to control insect pests like the aphid. In particular, secondary metabolites produced by entomopathogenic fungi are being studied as insecticidal substances. This study evaluated the insecticidal activity and characteristics of insecticidal substances in culture filtrate against aphids to confirm the insecticidal substances produced by Metarhizium pinghaense 15R. After treating cotton aphids with the culture filtrate, strong insecticidal activity (100 % insect mortality) was observed on the third day of treatment, and it was confirmed that insecticidal substances in the culture filtrate had high thermal stability. In addition, the physicochemical properties of insecticidal substances in the culture filtrate were evaluated, and it was determined that the insecticidal substances were hydrophobic, with small molecules of 3 kDa or less. Based on these results, it was suspected that the insecticidal substances in the culture filtrate were destruxins (DTXs), which are representative secondary metabolites of Metarhizium known to have insecticidal activity. Through HPLC (High Performance Liquid Chromatography) analysis, the presence of DTX A and B was found in all active samples, and it was confirmed that the insecticidal substances of M. pinghaense 15R were DTXs. This study provides primary data for developing biochemical crop protection agents using these insecticidal substances, and we suggest the potential of using insecticidal substances of entomopathogenic fungi as biochemical pesticides in Korea.     

Role of fragmentation activity in cellulose hydrolysis

Most studies of cellulose hydrolysis have been carried out on three components of the cellulolytic systems, viz, endoglucanases, exoglucanases, and cellobiases. Little attention has been paid to the fragmentation activity of certain cellulolytic systems. We have noticed that despite being a more powerful degrader of modified cellulose (CMC), the 7-day grown culture filtrate of Myrothecium verrucaria was less effective than that of Trichoderma reesei at degrading pure unmodified cellulose. Scanning electron microscopy imaging showed that one distinguishing feature of the latter is its ability to fragment (macerate) the cellulose. Cellulose particle size decreased with time as it was incubated in the culture filtrate of T. reesei at 37 °C. This was used as a pre-treatment. Pre-treated cellulose was then washed and incubated with fresh T. reesei or M. verrucaria culture filtrates. Pre-treatment increased liberation of reducing sugars during subsequent incubation of cellulose in T. reesei culture filtrate but not in subsequent incubation in M. verrucaria culture filtrate. It was hypothesized that fragmentation activity of the pre-treatment opened up attack sites for further hydrolysis, but these were not available for attack by other enzyme systems.     

Piriformospora indica, a Cultivable Plant-Growth-Promoting Root Endophyte

Piriformospora indica (Hymenomycetes, Basidiomycota) is a newly described cultivable endophyte that colonizes roots. Inoculation with the fungus and application of fungal culture filtrate promotes plant growth and biomass production. Due to its ease of culture, this fungus provides a model organism for the study of beneficial plant-microbe interactions and a new tool for improving plant production systems.     

Cultivation of syntrophic anaerobic bacteria in membrane-separated culture devices

A dialysis culture device was used for growth of syntrophic fatty acid-oxidizing and ethanol-oxidizing anaerobic bacteria. A pure culture of the fatty acid oxidizer Clostridium bryantii was grown inside dialysis tubing which was surrounded by a pure culture of Desulfovibrio vulgaris. The same apparatus was used for the syntrophic cultivation of Pelobacter acetylenicus and Acetobacterium woodii with ethanol as substrate. In both cases, substrate degradation and product formation were about half as fast as with the homogeneously mixed control cultures. In the compartment of the hydrogen producer, the concentration of free hydrogen during syntrophic ethanol degradation was about 10 times as high as in that of the hydrogen utilizer, whereas the homogeneously mixed culture exhibited an intermediate hydrogen partial pressure.     

Improved Succinic Acid Production in the Anaerobic Culture of an Escherichia coli pflB ldhA Double Mutant as a Result of Enhanced Anaplerotic Activities in the Preceding Aerobic Culture

Escherichia coli NZN111 is a pflB ldhA double mutant which loses its ability to ferment glucose anaerobically due to redox imbalance. In this study, two-stage culture of NZN111 was carried out for succinic acid production. It was found that when NZN111 was aerobically cultured on acetate, it regained the ability to ferment glucose with succinic acid as the major product in subsequent anaerobic culture. In two-stage culture carried out in flasks, succinic acid was produced at a level of 11.26 g/liter from 13.4 g/liter of glucose with a succinic acid yield of 1.28 mol/mol glucose and a productivity of 1.13 g/liter·h in the anaerobic stage. Analyses of key enzyme activities revealed that the activities of isocitrate lyase, malate dehydrogenase, malic enzyme, and phosphoenolpyruvate (PEP) carboxykinase were greatly enhanced while those of pyruvate kinase and PEP carboxylase were reduced in the acetate-grown cells. The two-stage culture was also performed in a 5-liter fermentor without separating the acetate-grown NZN111 cells from spent medium. The overall yield and concentration of succinic acid reached 1.13 mol/mol glucose and 28.2 g/liter, respectively, but the productivity of succinic acid in the anaerobic stage dropped to 0.7 g/liter·h due to cell autolysis and reduced anaplerotic activities. The results indicate the great potential to take advantage of cellular regulation mechanisms for improvement of succinic acid production by a metabolically engineered E. coli strain.