The flotation characteristics of Bacillus cells and spores]

Variations in hydrophobicity of the surface of bacillary cells and their capacity to flotation in the process of batch cultivation have been studied. It is shown that hydrophobicity of the cell surface increases in the course of batch cultivation of Bacillus thuringiensis, B. licheniformis and B. megaterium. Hydrophobicity of spores of the mentioned cultures is considerably higher than that of the vegetative cells. The increase of hydrophobicity of bacillary cells positively correlated with their capacity to flotation. That is why the use of flotation for the age fractionation of bacillary cells is possible: spores are concentrated in the foam while vegetative cells remain in the culture liquid.     

The use of sewage sludge and horticultural waste to develop artificial soil for plant cultivation in Singapore

Greenhouse pot experiments were performed with Ipomoea aquatica (Kang Kong) to evaluate artificial soil produced from poor fertility subsoil, horticultural compost, and sewage sludge. The addition of horticultural compost and sewage sludge to subsoil substantially improved plant growth, improved the physical properties of subsoil and enriched subsoil by essential nutrients for plants. The effect was enhanced when the two ingredients were added to subsoil together. The highest yield of biomass of I. aquatica was observed in artificial soil prepared by mixing subsoil with 4% (wet weight/wet weight) of horticultural compost and 2% (dry weight/wet weight) of sewage sludge. The contents of heavy metals in plants, grown in the artificial soil, were significantly lower than toxic levels. The artificial soil could be recommended for urban landscaping and gardening in Singapore.     

Starter culture of <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> strain B for aerobic granulation

Aggregation of bacterial cells is used in formation of microbial granules. Aerobically grown microbial granules can be used as the bio-agents in the treatment of wastewater. However, there are problems with start up of microbial granulation and biosafety of this process. Aim of this research was selection and testing of safe microbial strain with high cell aggregation ability to shorten period of microbial granules formation. Five bacterial strains with cell aggregation index higher than 50% have been isolated from the granules. Strain of Pseudomonas veronii species was considered as most probably safe starter culture for granulation because other strains belonged to the species known as human pathogens. The microbial granules were formed after 3 days of cultivation in case when P. veronii strain B was applied to start-up aerobic granulation process using model wastewater. The granules were produced from activated sludge after 9 days of cultivation. Microbial aggregates produced from starter culture of P. veronii strain B were more compact (sludge volume index was 70 ml/g) than those produced from activated sludge (sludge volume index was 106 ml/g). It is a first proof that application of selected safe starter pure culture with high cell aggregation ability can accelerate and enhance formation of microbial granules.     

Study of asynchronous and synchronous yeast cultures using flow cytofluorometry]

Distribution of Saccharomyces cerevisiae, Candida boidinii and Candida tropicalis cells according to DNA content was investigated using laser flow cytofluorometry. Cells distribution curves according to DNA content possessed two maxima in case the sample belonged to the exponential phase of the asynchronous batch culture, or one maximum in case the sample was from the stationary phase of growth. In synchronous cultures variations of cells distribution curves according to DNA content (age structure of the population) were demonstrated and the curves with one maximum and plateau were observed.     

Age specificity of the interaction of Bacillus cells with a liquid-gas interface]

The age specificity of the bacilli cells with liquid--gas interface was studied using flow cytofluorometry. This interactions is dependent on the position of the cell in the division cycle and not on the position of the cell in the DNA replication cycle.     

Quantification of methanogens by fluorescence in situ hybridization with oligonucleotide probe

To monitor anaerobic environmental engineering system, new method of quantification for methanogens was tested. It is based on the measurement of specific binding (hybridization) of 16S rRNA-targeted oligonucleotide probe Arc915, performed by fluorescence in situ hybridization (FISH) and quantified by fluorescence spectrometry. Average specific binding of Arc915 probe was 13.4±0.5 amol/cell of autofluorescent methanogens. It was 14.3, 13.3, and 12.9 amol/cell at the log phase, at stationary phase and at the period of cell lysis of batch culture, respectively. Specific binding of Arc915 probe per 1 ml of microbial sludge suspension from anaerobic digester linearly correlated with concentration of autofluorescent cells of methanogens. Coefficient of correlation was 0.95. Specific binding of oligonucleotide probe Arc915 can be used for the comparative estimation of methanogens during anaerobic digestion of organic waste. Specific binding of Arc915 probe was linear function of anaerobic sludge concentration when it was between 1.4 and 14.0 mg/ml. Accuracy of the measurements in this region was from 5 to 12%.     

Anaerobic digestion of food waste in a hybrid anaerobic solid–liquid system with leachate recirculation in an acidogenic reactor

Recirculation of the leachate in the acidogenic reactor was proposed to enhance anaerobic digestion of food waste in the hybrid anaerobic solid–liquid (HASL) system. Recirculation of the leachate in the acidogenic reactor provided better conditions for extraction of organic matter from the treated food waste and buffering capacity to prevent excessive acidification in the acidogenic reactor. It ensured faster supply of nutrients in the methanogenic reactor in experiment. The highest dissolved COD and VFA concentrations in the leachate from the acidogenic reactor were reached for shorter time and were 16,670 mg/l and 9450 mg/l in control and 18,614 mg/l and 11,094 mg/l in experiment, respectively. Recycling of the leachate in the acidogenic reactor intensified anaerobic digestion of food waste and diminished time needed to produce the same quantity of methane by 40% in comparison with anaerobic digestion of food waste without recirculation.     

Hydrophobic interactions within biofilms of nitrifying and denitrifying bacteria in biofilters

Hydrophobicity of the solid surface and microbial cell surface is important factor for the development of biofilms applied in bioengineering systems. An adsorption of phenanthrene was used for analysis of the hydrophobicity of support fibers and bacterial cell surfaces within the biofilter of wastewater. The adsorption of phenanthrene was measured by synchronous fluorescence spectrometry. Cell surface hydrophobicity does not depend on the fixation procedure, pH of microbial suspension, and has no clear correlation with an adherence of the cells to hexadecane droplets. Notwithstanding high hydrophobicity of bacterial cells, the hydrophobicity of intact biofilm is determined by the hydrophobicity of the support fibers. New indexes were proposed to evaluate the reactor performance related with hydrophobic interactions within the biofilm. These indexes showed that significant share of hydrophobic sites within the nitrifying biofilm is protected from the hydrophobic interactions between the cells and environment.     

Use of the level of G+C n DNA for studying the molecular phylogeny o f methanogenic archaebacteria

A new method for theoretical analysis of the molecular phylogeny of bacteria, successfully applied earlier to nitrifying bacteria, was used to study the molecular phylogeny of methanogenic archaebacteria. The group studied included Methanococcus igneus, Methanococcus vannielii, Methanothermus fervidus, Methanolobus tindarius, Methanobacterium formicicum, Methanosarcina barkeri, Methanobacterium thermoformicicum, Methanoplanus limicola, Methanospirillum hungatei, and Methanobacterium thermoautotrophicum. Based on the hypothesis that direct linear regression always exists between evolutionary changes in the DNA G + C content and the primary structure of rRNA, the branching order of the phylogenetic tree of methanogenic archaebacteria was determined. For this tree, the values of the evolutionary distance between 16S rRNA primary structures Ei and the values of the G + C evolutionary distance P(i) exhibited a correlation coefficient 0.78. Thus, the DNA G + C content is not only an important taxonomic characteristics but also provides information helpful for the determination of the branching order of phylogenetic trees constructed based on 16S rRNA primary structures.     

Microbiological monitoring in the biodegradation of sewage sludge and food waste

AIM: To study the microbiology of intensive, in-vessel biodegradation of a mixture of sewage sludge and vegetable food waste. METHODS AND RESULTS: The biodegradation was performed in a closed reactor with the addition of a starter culture of Bacillus thermoamylovorans SW25 under conditions of controlled aeration, stirring, pH and temperature (60 degrees C). The content of viable bacterial cells, determined by flow cytometry, increased from 5 x 108 g-1 of dry matter to 61 x 108 g-1 for 6 days of the process and then dropped to the initial value at the end of the process. The reductions of organic matter, 16S rRNA of methanogens and coenzyme F420 fluorescence during 10 days of the treatment were 67, 54 and 87% of the initial values, respectively. The biodegradability of the organic matter decreased during the 10 days of the treatment from 3.8 to 1.3 mg CO2 g-1 of organic matter per day. The treatment of sewage sludge and food waste at 60 degrees C did not remove enterobacteria, which are the agents of intestinal infections, from the material. The percentage of viable enterobacterial cells, determined by fluorescent in situ hybridization (FISH) with Enterobacteriaceae-specific oligonucleotide probe and flow cytometry, varied from 1 to 14% of the viable bacterial cells. CONCLUSIONS: The mixture of sewage sludge and food waste can be degraded by the aerobic thermophilic bacteria; the starter culture of Bacillus thermoamylovorans SW25 can be used to perform this process; and enterobacteria can survive under treatment of sewage sludge and food waste at 60 degrees C for 13 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that FISH with an oligonucleotide probe can be used to study not only the growth but also the degradation of biomass. Obtained results could be used to design the bioconversion of sewage sludge and food waste into organic fertilizer.