Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions

A variety of white-rot fungi can oxidize textile dyes under sterile conditions; however, an important consideration for their use in treating wastewater containing textile dyes is whether similar degrees of treatment can be achieved under non-sterile conditions. Four strategies were investigated for their potential in optimizing the use of the fungus Trametes versicolor in non-sterile culture for treating wastewater containing the diazo textile dye C.I. Reactive Black 5 (RB5). Three strategies with suspended culture were designed to increase the decolorization activity in suspended culture from a given amount of T. versicolor inoculum based on its tolerance of low pH (pH reduction in medium), production of extracellular enzymes (use of suspended enzymes alone), and its ability to produce enzymes independent of growth (nitrogen limitation in medium). The results showed that reduction of the medium pH to 3 did not suppress bacterial growth, while enzyme production by T. versicolor ceased. The use of the extracellular enzymes alone would allow the decoupling of the process of fungal growth from wastewater treatment; however, the enzyme activity of an enzyme suspension decreased rapidly under non-sterile conditions. The strategy of limiting nitrogen in the medium to suppress bacterial growth has potential together with the fourth strategy, the cultivation of fungi on organic solids to produce inocula for a decolorization process under non-sterile conditions. A high degree of decolorization of RB5 under non-sterile conditions was achieved with T. versicolor grown on grains as sole substrate. The rate of decolorization was dependent on the amount of fungal inoculum used.     

Bacteriolytic enzymes from Staphylococcus aureus. Purification of an endo-β-N-acetylglucosaminidase

On cultivation of Staphylococcus aureus in a complex liquid medium, bacteriolytic activity is found extracellularly. The maximal amount was found at the end of the exponential growth phase in batch culture, but in continuous culture run under similar conditions the yield was doubled. Isoelectric focusing of dialysed crude culture supernatants showed that the bacteriolytic activity of all four strains studied (M18, 524, Wood 46 and Duncan) was heterogeneous. The most alkaline peak of activity (isoelectric point 9.5±0.1) was assayed against Micrococcus lysodeikticus turbidimetrically. This bacteriolytic activity was purified more than 70-fold after continuous dialysis by adsorption on CM-Sephadex, precipitation with ethanol, heat purification, isoelectric focusing and Sephadex G-100 chromatography. The purified enzyme (isoelectric point 9.6±0.1) was found to give a single band on polyacrylamide-gel and cellulose acetate electrophoresis and was devoid of all 14 staphylococcal enzymes and toxins assayed for. The molecular weight is 70000±5000 as estimated by Sephadex G-100 and G-200 chromatography. The marked instability of the partially and highly purified enzyme was investigated. The mode of action and some properties of this enzyme are given in the following papers (Wadström & Hisatsune, 1970; Wadström, 1970). These results indicate that this extracellular enzyme which is produced by several strains of S. aureus is not a `lysozyme' (endo-β-N-acetylmuramidase) as previously suggested, but an endo-β-N-acetylglucosaminidase.     

Autolysis of Lactococci: Detection of Lytic Enzymes by Polyacrylamide Gel Electrophoresis and Characterization in Buffer Systems

Lactococcal strains were screened for bacteriolytic activity against Micrococcus luteus cells, lactococcal cells, and cell walls. Thirty strains were screened for bacteriolytic activity against cells and cell walls incorporated into agar medium. Enzymes from all strains hydrolyzed the substrates; however, the activity against Micrococcus cells was much higher than against Lactococcus cells or cell walls. Electrophoretic profiles of bacteriolytic activities of culture supernatants, sodium dodecyl sulfate-treated cell extracts, cell wall fractions, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing M. luteus cells or lactococcal cell walls as the substrate. The 22 strains tested contained two to five lytic bands in the culture supernatant, ranging in size between 32 and 53 kDa. The cell extracts, the sodium dodecyl sulfate-treated cell extracts, and the cell wall fractions revealed two lytic bands of 47 and 53 kDa. Effects of external factors on autolysis of some strains were determined in buffer systems. Optimal autolysis was observed in the exponential growth phase at pH 6.0 to 7.5 and at a temperature of 30(deg)C. Two of three strains tested seemed to contain a glycosidase, and all three strains contained an N-acetylmuramyl-l-alanine amidase or an endopeptidase.     

Role of hydrolytic enzymes and oxidative stress in autolysis and morphology of <Emphasis Type="Italic">Blakeslea trispora</Emphasis> during β-carotene production in submerged fermentation

The role of hydrolytic enzymes (proteases and chitinase) and oxidative stress in the autolysis and morphology of Blakeslea trispora during β-carotene production from a chemically defined medium in shake flask culture was investigated. The process of cellular autolysis was studied by measuring the changes in biomass dry weight, pH, concentration of β-carotene, specific activity of the hydrolytic enzymes and micromorphology of the fungus using a computerized image analysis system. In addition, the phenomenon of autolysis was associated with high concentrations of reactive oxygen species (ROS). The accumulation of ROS produced during fermentation causes oxidative stress in B. trispora. Oxidative stress was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). The profile of the specific activities of the above enzymes appeared to correlate with the oxidative stress of the fungus. The high activities of CAT and SOD showed that B. trispora is found under oxidative stress during β-carotene production. The culture began to show signs of autolysis nearly in the growth phase and autolysis increased significantly during the production phase. The morphological differentiation of the fungus was a result of the degradation of the cell membrane by hydrolytic enzymes and oxidative stress. Increased β-carotene production is correlated with intense autolysis of clumps, which has as a consequence the increase of the freely dispersed mycelia.     

Variants of Myxococcus virescens Exhibiting Dispersed Growth. Growth and Production of Extracellular Enzymes and Slime

The growth of two strains of Myxococcus virescens exhibiting dispersed growth was followed in casamino acids (N III-C) media and casitone media. The changes in optical density, pH, pigmentation as well as the secretion of bacteriolytic and proteolytic enzymes, DNA and polysaccharides during growth were recorded. In both media the bacteria grew exponentially with a generation time of 4 (casitone) and 20 hours (N III-C) respectively. The maximal cell mass was about 4 times higher in casitone than in casamino acids media. The amounts of bacteriolytic enzymes produced by the two strains in N III-C medium were different but in casitone medium they were about equal and considerably higher. The maximal values of proteolytic enzymes were about the same in both media and always occurred later than the bacteriolytic maxima. Both activity peaks appeared before the phase of decline. The polysaccharide production reached a maximum during the stationary growth phase in both media. A higher value was reached during growth in casitone medium than in N III-C medium. During the phase of decline a second increase of polysaccharide in the medium appeared. No DNA could be detected in the cell-free solutions until the beginning of the phase of decline.     

The cellular location of proteolytic enzymes ofBacillus intermedius

The activities of proteinases in the culture fluid and cellular fractions ofBacillus intermedius 3–19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. The production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2-to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of C0Cl₂ (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted.     

Secretion of bacteriolytic endopeptidase L5 of Lysobacter sp. XL1 into the medium by means of outer membrane vesicles

The Gram-negative bacterium Lysobacter sp. XL1 secretes various proteins, including bacteriolytic enzymes (L1-L5), into the culture medium. These proteins are able to degrade Gram-positive bacteria. The mechanism of secretion of extracellular proteins by Lysobacter sp. XL1 has not been studied hitherto. Electron microscopic investigations revealed the phenomenon of the formation of extracellular vesicles by Lysobacter sp. XL1. These vesicles contained components of the Lysobacter sp. XL1 outer membrane, and demonstrated bacteriolytic activity against Gram-positive and Gram-negative bacteria: Staphylococcus aureus 209-P and Erwinia marcescens EC1, respectively. Western blotting analysis with antibodies to homologous bacteriolytic endopeptidases L1 and L5 showed that endopeptidase L5 was secreted into the culture medium by means of vesicles, unlike its homolog, endopeptidase L1. When inside the vesicles, endopeptidase L5 actively lysed the Gram-negative bacterium Erwinia marcescens; outside the vesicles, it lost this ability. The secretion of bacteriolytic endopeptidase L5 through the outer membrane vesicles is of great biological significance: because of this ability, Lysobacter sp. XL1 can compete in nature with both Gram-positive and Gram-negative bacteria.     

Localization of proteolytic enzymes in cells of Bacillus intermedius

The activities of proteinases in the culture fluid and cellular fractions of Bacillus intermedius 3-19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. Production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2- to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of CoCl2 (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted.     

Alterations in intracellular and extracellular activities of antioxidant enzymes during suspension culture of sweetpotato

Cultured plant cells are a good system for the study of antioxidant mechanisms and for the mass production of antioxidants, because they can be grown under conditions of high oxidative stress. Alterations in the intracellular and extracellular activities of three antioxidant enzymes, superoxide dismutase (SOD), guaiacol-type peroxidase (POD), and glutathione peroxidase (GPX), were investigated in suspension cultures of sweetpotato (Ipomoea batatas) during cell growth. Intracellular SOD activities (units/mg protein) at 15 days after subculture (DAS) and 30 DAS were 10 and 20 times higher, respectively, compared with the SOD activity at 1 DAS, whereas intracellular specific POD and GPX activities did not significantly increase until after 15 DAS, when they rapidly increased. The extracellular activities of the three enzymes in culture medium were much higher than were the intracellular activities. The change in extracellular SOD activity was similar to that of extracellular GPX during cell growth. Those activities showed high levels until 5 DAS and then significantly decreased. Extracellular POD activity had an almost constant level regardless of the cell growth stage. In addition, intracellular SOD and POD isozymes were quite different from those isozymes in the culture medium. The changes in SOD and POD isozymes observed here suggest that different isozymes might modulate the levels of reactive oxygen intermediates during cell growth. Characterization of extracellular antioxidant enzymes discovered here would provide a new understanding for defense mechanism in plants.     

Production and detection of muramidase and acetylglucosaminidase from Agaricus bisporus

The production and regulation of extracellular bacteriolytic enzymes of Agaricus bisporus are being studied to understand better the nutrition of this fungus and to identify factors that regulate the selectivity of mushroom compost as a growth medium. Both muramidase (EC.3.2.1.17) and N -acetyl-β- D -glucosaminidase (β-GlcNAcase, EC.3.2.1.30) have been detected in liquid cultures of A. bisporus , and in cultures fruiting in sterile and non-sterile compost. A turbidometric assay, based on the decrease in optical density of suspended Bacillus subtilis bacterial cell walls, was used to measure muramidase production by A. bisporus . A colorimetric assay was used to measure β-GlcNAcase. Both bacteriolytic enzyme activities were produced on a range of sole carbon sources, including killed freeze-dried B. subtilis cells. Muramidase activity was highest in axenic compost cultures. Bacteriolytic enzyme activity peaked as the first group of fruit bodies was harvested in both sterile and non-sterile compost.     

Effect of carbon source and dissolved oxygen level on cell growth and pullulanase production byBacillus stearothermophilus G-82

Cell growth and extracellular pullulanase production ofBacillus stearothermophilus G-82 were investigated in batch culture using a defined medium with glucose, maltose, pullulan or amylopectin as carbon source. Maximum enzyme activity was with pullulan or amylopectin. Cell growth in batch culture was better under oxygen unlimited conditions, while higher total and specific enzyme activities, using pullulan or amylopectin, were obtained in oxygen-limited conditions. Enzyme accumulation took place in the late growth phase. The highest enzyme production of 300 U/I was reached when pullulan was used as carbon source in conditions of oxygen limitation.     

Derepression of beta-1,3-glucanases in Penicillium italicum: localization of the various enzymes and correlation with cell wall glucan mobilization and autolysis.

The localization of the derepressible beta-1,3-glucanases of Penicillium italicum and the cell wall autolysis under conditions of beta-1,3-glucanase derepression (24 h in a low-glucose medium) were studied. About 15% of the total activity was secreted into the culture medium during the 24-h period and consisted of similar amounts of each of the three beta-1,3-glucanases (I, II, III) produced by this species. Treatment of derepressed mycelia with periplasmic enzyme-inactivating agents resulted in a loss of 45% of the mycelium-bound beta-1,3-glucanase. Analysis of periplasmic enzymes solubilized by 2 M NaCl or by autolysis of isolated cell walls revealed that only beta-1,3-glucanases II and III were bound to the cell wall. These two enzymes were capable of releasing in vitro reducing sugars from cell walls, whereas beta-1,3-glucanase I was not. In addition, the autolytic activity of cell walls isolated from derepressed mycelium was greater than that of cell walls isolated from repressed mycelium. The incubation of the fungus in the low-glucose medium also resulted in the in vivo mobilization of 34% of the cell wall beta-1,3-glucan, and this mobilization was fully prevented by cycloheximide, which also blocked derepression of beta-1,3-glucanases. Derepression of beta-1,3-glucanase seems to be coupled to the mobilization of cell wall glucan.     

Effect of physiological conditions on the autolysis ofStaphylococcus aureus strains

The effect of physiological conditions on autolysis and autolytic activity in various strains ofStaphylococcus aureus was determined. The rate of whole cell autolysis ofS. aureus was growth phase dependent and a maximum rate was observed in early stationary phase cultures. However, the autolysins extracted by the freeze-thaw method (cell-wall bound autolytic activity) did not show any significant increase in activity. The addition of NaCl to the growth medium enhanced the rate of autolysis with the highest rate being displayed by cultures grown in 1.5 M NaCl. However, lower autolytic activity was found in the freeze-thaw extracts of cultures grown at higher concentrations of NaCl. The rate of autolysis of cultures grown at 30°C was higher than cultures grown at 37 or 43°C. Thus, the rate of autolysis seems to be independent of the bacterial growth rate. Cultures grown in slightly acidic conditions showed a faster rate of autolysis compared to cultures grown under alkaline conditions. SDS-polyacrylamide gel containing 0.2% crude cell-wall ofS. aureus did not show any obvious correlation with the appearance of any particular lytic band in the zymogram to autolytic activity or rate of autolysis of cultures grown under various environmental conditions. A nonhemolytic phenotype, mutations in the accessory gene regulator, and lysogeny (phages ø11, ø12, ø13) had no obvious effect either on the rate of autolysis or on the pattern of lytic bands in the zymograms.     

Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides.

Leuconostoc mesenteroides NRRL B-512(F) was grown in continuous culture under conditions of energy-limited growth. The extracellular enzyme dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5), was not detected in glucose- or maltose-limited cultures. Under conditions of sucrose-limited growth, the enzyme activity of the cell-free culture supernatant increased with increasing dilution rate only after the critical concentration of enzyme inducer (sucrose) in the chemostat had been achieved. The appearance of fructose in the effluent of the sucrose-limited chemostat at higher dilution rates indicated that sucrose was being diverted to dextran biosynthesis. The competition between bacteria and extracellular enzyme for the common substrate sucrose represents an inefficiency in the system of enzyme production. Dextransucrase was isolated from the cell-free culture supernatant by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme preparation exhibited both dextran biosynthetic activity and an invertase-like activity. The biosynthetic efficiency was increased by decreasing the temperature from 30 to 10 degrees C. The enzyme was irreversibly denatured by prolonged incubation in the absence of Ca2+.     

Effect of regulators on bacterial autolysis

The aim of this work was to study the effect of autolysis regulators (the fraction of microbial teichoic acids) on the rate of autolysis and the activity of bacterial extracellular lytic enzymes. The regulators of autolysis isolated from 23 cultures belonging to 10 microbial species regulated the rate of autolysis in Bacillus, E. coli and Streptococcus lactis. The regulators either activated or inhibited autolysis depending on the substrate (of a bacterium to be subjected to autolysis). The quantitative dependence of the autolysis rate on the regulator concentration was specific for each pair 'regulator--substrate'. The regulatory properties of the fraction of teichoic acids varied depending on the age of a culture from which they had been isolated. The regulators of autolysis, with an exception of the preparation from E. coli, inhibited the activity of B. subtilis extracellular lytic enzymes in the course of their action on E. coli cells. The possibility for using the regulators of autolysis in microbiological processes is discussed.     

Chitinolytic activity in the autolysis of Aspergillus nidulans

Abstract Chitinolytic activity in filtrates of Aspergillus nidulans cultures was studied at the start of the autolysis (maximum dry weight of mycelium) and during autolysis in 24 different media. During the growth the chitinolytic activity was induced only by the presence of ascorbic acid or colloidal chitin in the medium. During autolysis an increasing chitinolytic activity was observed with the incubation time in all the conditions, and synthesis of a β - N -acetylgucosaminidase and endochitinase was detected. The possible induction of these enzymes during A. nidulans autolysis is established.     

Continuous production of extracellular protease by Bacillus subtilis in a two-stage fermentor

Growth and protease production of Bacillus subtilis in semisynthetic and synthetic media were studied in batch culture and in a two-stage, laboratory scale, continuous fermentor. The amount, of extracellular protease production was measured under specific growth conditions in both stages of the ferment or. At the dilution rates employed, the cells in the first stage of the ferment or produced negligible quantities of protease, and the culture primarily functioned as a continuous inoculum for the second stage of the fermentor. The culture effluent from the second stage of the fermentor contained extracellular protease, on the average, equal to 60 per cent, of the activity that had been found in the supernatant of a 48-hr batch culture grown in a medium having the same composition as that in the continuous fermentor. Extracellular protease was produced in semisynthetic medium by B. subtilis in the two-stage fermentor for as long as 20 days without culture degeneration. Additional studies indicated that continuous protease production could also be achieved in a synthetic medium. The RNA/ protein ratios of cells grown in semisynthetic medium in batch culture and in each stage of the two-stage fermentor were examined. There was a positive correlation between the amount of protease produced by the cells and their RNA/ protein ratio. Techniques employed for continuous production of protease by B. subtilis and the potential use of the method for investigating the control of secondary metabolite synthesis are discussed.     

Production of cell-bound and vesicle-associated trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase by Bacteroides gingivalis strain W50

Bacteroides gingivalis strain W50 was grown in batch and continuous culture on complex medium with haemin. In batch culture, cell-bound levels of trypsin-like protease (EC 3.4.21.4), alkaline phosphatase (EC 3.1.3.1) and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) increased during the exponential phase of growth. These enzyme activities were also detected in extracellular vesicles and in extracellular soluble forms in the supernatant fluid, but in lower amounts per unit biomass compared to cell-bound levels. In continuous culture, at high relative growth rates (0.7-0.9 murel), the highest proportions of enzyme activities were cell-bound. In contrast, at low relative growth rates (0.1-0.2 murel), highest enzyme levels were detected in the extracellular vesicle fraction. Levels of extracellular soluble enzymes were always low compared to cell-bound or extracellular vesicle levels, but were highest at low relative growth rates. All three enzymes appeared to be relatively stable in their soluble forms. Vesicle production appeared to be associated with actively growing cells but was influenced by growth rate. The results are consistent with the hypothesis that cell-bound 'periplasmic' enzymes are encapsulated into vesicles which are subsequently released by the cells. Therefore, levels of total extracellular enzyme (extracellular vesicle plus extracellular soluble) may depend on the rate of vesicle formation superimposed on the rates of production of 'periplasmic' enzymes in the cell.     

Production and characterization of the agarase ofCytophaga flevensis

Cytophaga flevensis produced an inducible agarase which was extracellular under most conditions tested. The effect of cultural conditions on the production of enzyme was studied in batch and continuous culture. In batch culture, production was optimal whenCytophaga flevensis was incubated at 20C in a mineral medium with agar as the sole carbon source and ammonium nitrate as the nitrogen source at an initial pH of 6.6–7.0. The enzyme appeared to be subject to catabolite repression, since its synthesis was repressed when glucose was added to the medium in batch culture. Furthermore, in continuous culture, enzyme production decreased with increasing growth rate. Extracellular agarase was partially purified and the enzyme preparation obtained was very stable. The enzyme has a molecular weight of 26000 daltons. It is a β-agarase which is highly specific for polysaccharides containing neoagarobiose units. The final products of hydrolysis of agarose by the endo-acting enzyme were neoagarotetraose and neoagarobiose. Optimal conditions for its activity were pH 6.3 and 30C. When agarose was used as a substrate, an apparent temperature optimum of 35C was found, due to gelling of the substrate during the assay procedure.     

Involvement of microbial alkyl hydroxybenzenes in the regulation of autolytic degradation of yeast cells

A comparative study was performed of the processes of autolytic degradation of the cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe under conditions simulating the phase of cell death in microbial cultures: (1) during autolysis induced by oleic acid, which is the chemical analogue of factors d2 (autolysis autoinducer), (2) under the effect of extracellular microbial proteinases (enzymatic lysis), and (3) under concomitant effect of the enzymes of the endogenous autolytic complex and exogenous proteinases (heterolysis). Regulatory mechanisms controlling the rate and profundity of autolysis were elucidated, relying on the stabilization of hydrolytic enzymes and enhancement of their activity in their complexes with a chemical analogue of microbial autoregulatory factors d1, which belong to alkylhydroxybenzenes and fulfil functions of chemical chaperons. The changes in the activity of proteinases and enzymes of the autolytic complex were shown to be dependent on the concentration of the analogue at the moment of complex formation.