Dimitrina Spasova

Rhamnolipid–Biosurfactant Permeabilizing Effects on Gram-Positive and Gram-Negative Bacterial Strains

The potential of biosurfactant PS to permeabilize bacterial cells of Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis on growing (in vivo) and resting (in vitro) cells was studied. Biosurfactant was shown to have a neutral or detrimental effect on the growth of Gram-positive strains, and this was dependent on the surfactant concentration. The growth of Gram-negative strains was not influenced by the presence of biosurfactant in the media. Cell permeabilization with biosurfactant PS was shown to be more effective with B. subtilis resting cells than with Pseudomonas aeruginosa. Scanning-electron microscopy observations showed that the biosurfactant PS did not exert a disruptive action on resting cells such that it was detrimental to the effect on growing cells of B. subtilis. Low critical micelle concentrations, tender action on nongrowing cells, and neutral effects on the growth of microbial strains at low surfactant concentrations make biosurfactant PS a potential candidate for application in different industrial fields, in environmental bioremediation, and in biomedicine.

Thermostable exo-inulinase production by semicontinuous cultivation of membrane-immobilized Bacillus sp. 11 cells

Abstract Growing cells of thermophilic Bacillus sp. 11, producer of a thermostable exo-inulinase, were immobilized on formaldehyde-activated polysulphone membranes. The biocatalysts obtained showed 1.5–2 fold higher enzyme yields (inulinase and invertase activities) than those of free cells. Active cell growth and no alterations of the membrane structure were observed by scanning electron microscopy (SEM) after 10 days (five cycles) repeated batch cultivation. The residual enzyme activities of the biocatalysts were 60–90% of their initial activities at the end of fifth run of semicontinuous cultivation.

Immobilization of Bacillus licheniformis cells, producers of thermostable α-amylase, on polymer membranes

Cells of Bacillus licheniformis 44MB82-G immobilized on different polymer membranes were used for production of thermostable α-amylase. The α-amylase yields of the membrane-immobilized cells were affected by the reactive chemical groups of the carriers and the spacer size. Formaldehyde-activated polysulphone membranes (PS-FA) were the most suitable for effective immobilization. The highest amylase yield (62% increase of the control) and operational stability (97% residual activity after 480 h repeated batch cultivation) were obtained with this system. This was confirmed by scanning electron micrographs. An additional increase of α-amylase production by PS-FA-membrane immobilized cells was achieved in a fluidized-bed reactor.

Agar gel immobilization ofBacillus brevis cells for production of thermostable α-amylase

Growing cells of a thermophilic strain ofBacillus brevis, producer of thermostable α-amylase, were immobilized by entrapment in agar gel. Optimum immobilization conditions for effective α-amylase production in batch fermentations were established (gel concentration 3%, initial biomass concentration in the gel 0.8% (W/V), and preculture age—late exponential phase). The dynamics of α-amylase synthesis by the biocatalysts obtained under the optimal conditions was compared with that of free cells and the operational stability of the biocatalysts was studied in semicontinuous cultivation experiments. Maximum α-amylase yields (252% of the control) were achieved after the second cycle of cultivation. Scanning electron microscopy was used to characterize the bacteria entrapped in agar gel.

Acid phosphatase distribution and localization in the fungus Humicola lutea

Acid phosphatase activities in a culture liquid and mycelial extract were studied in submerged cultures of the filamentous fungus Humicola lutea 120-5 in casein-containing media with and without inorganic phosphate (Pi). The Pi-repressible influence on the phosphatase formation was demonstrated. Significant changes in the distribution of acid phosphatase between the mycelial extract and culture liquid were observed at the transition of the strain from exponential to stationary phase. Some differences in the cytochemical localization of phosphatase in dependence of Pi in the media and the role of the enzyme in the release of available phosphorus from the phosphoprotein casein for fungal growth were discussed.

Production and Properties of a Bacterial Thermostable Exo-inulinase

Abstract Enzyme production of newly isolated thermophilic inulin-degrading Bacillus sp. 11 strain was studied by batch cultivation in a fermentor. The achieved inulinase and invertase activi ties after a short growth time (4.25 h) were similar or higher compared to those reported for other mesophilic aerobic or anaerobic thermophilic bacterial producers and yeasts. The investigated enzyme belonged to the exo-type inulinases and splitted-off inulin, sucrose and raffinose. It could be used at temperatures above 65 °C and pH range 5.5-7.5. The obtained crude enzyme preparation possessed high thermostability. The residual inulinase and inver tase activities were 92-98% after pretreatment at 65 °C for 60 min in the presence of substrate inulin.

IMMOBILIZATION IN NANOMATRICES OF HUMICOLA LUTEA MYCELIUM FOR ALPHA-GALACTOSIDASE BIOSYNTHESIS IN LABORATORY AIR-LIFT BIOREACTOR

ABSTRACT The sol-gel synthesis of both hybrid nanomatrices containing tetraethylortosilicate (TEOS) as an inorganic precursor and lactic acid, or sepharose as an organic component was made. Crystal as well as surface morphology of the hybrids were investigated using different methods: X-ray diffraction, infrared spectra, BET-analysis and atomic force microscopy (AFM). The obtained nanomatrices were applicated for immobilization of the α-galactosidase producing fungal strain Humicola lutea 120–5. The semicontinuous cultivation was carried out in laboratory air-lift bioreactor. Maximal level of enzyme activity (1050–1130 U/l) that was reached in the third to fourth fermentation cycle using TEOS+40% lactic acid was higher than that obtained in the samples with TEOS+20% sepharose (660–770 U/l). The correlation between enzyme productivity and fungal development in the pore structure of the carriers was examined using scanning electron microscopy observation.

Examination of Humicola lutea Immobilized in Sol-Gel Matrices: Effective Source of α-Galactosidase

Abstract α-Galactosidase production by the fungus Humicola lutea 120-5 immobilized in a hybrid sol-gel matrix, consisting of tetraethylorthosilicate (TEOS) as a precursor and a mixture of polyethyleneglycol (PEG) and polyvinylalcohol (PVA), was investigated under semicontinuous shake flask cultivation and compared to the enzyme secretion by free cells. The influence of the carrier weight on the α-galactosidase biosynthesis in repeated batch experiments was followed. Best results were obtained with 2 g of the sol-gel particles per culture flask using 144-h runs. The growth behaviour of the immobilized mycelium during both the growth and productive phases was observed by scanning electron microscopy. The presence of abundant mycelial growth of intact hyphae correlated with a 2-fold higher enzyme activity compared to free cells. The obtained biocatalyst retained a high level of enzyme titer exceeding the activity of free cells during four cycles of operation (24 days). This result is confirmed by the micrographs showing the retained viability of the growing vegetative cells due to the protective role of the carrier.

Ultracytochemical localization of acid phosphatase in Humicola lutea conidia and mycelia.

Electron microscopic cytochemical procedures were used to determine the cellular location of acid phosphatase in the fungus Humicola lutea grown in casein-containing medium lacking in mineral orthophosphates. In our investigations acid phosphatase in nongerminating conidia was localized on the outer side of the cell wall, in the cell wall, and on the exterior surface of the plasma membrane. The reaction product of acid phosphatase in germinating conidia was seen in the outer wall layer while in young mycelium on the cell surface and in the exocellular space. The relationship between phosphatase activities localized in the cell wall and their role in the enzymatic degradation of the phosphoprotein casein providing available phosphates for cell growth is discussed.