Ozonation effects for excess sludge reduction on bacterial communities composition in a full-scale activated sludge plant for domestic wastewater treatment.

Activated sludge process is the most widely diffused system to treat wastewater to control the discharge of pollutants into the environment. Microorganisms are responsible for the removal of organic matter, nitrogen, phosphorous and other emerging contaminants. The environmental conditions of biological reactors significantly affects the ecology of the microbial community and, therefore, the performance of the treatment process. In the last years, ozone has been used to reduce excess sludge production by wastewater treatment plants (WWTPs), whose disposal represents one of the most relevant operational costs. The ozonation process has demonstrated to be a viable method to allow a consistent reduction in excess sludge. This study was carried out in a full-scale plant treating municipal wastewater in two parallel lines, one ozonated in the digestion tank and another used as a control. Bacterial communities of samples collected from both lines of digestion thanks were then compared to assess differences related to the ozonation treatment. Data were then analysed with terminal restriction fragment length polymorphism (T-RFLP) analysis on 16S rRNA gene. Differences between bacterial communities of both treated and untreated line appeared 2 weeks after the beginning of the treatment. Results demonstrated that ozonation treatment significantly affected the activated sludge in WWTP.

The effect of substituting energy crop with agricultural waste on the dynamics of bacterial communities in a two-stage anaerobic digester.

The replacement of energy crops with agricultural waste in biogas production through anaerobic digestion (AD) is both an environmentally sustainable and economically profitable strategy. However, the change of feeding mix in AD might result in nutrient imbalance or increase of the ammonium concentration, negatively affecting the activity of the microbes responsible for the process. In the present study the structure and dynamics of the bacterial communities of a full-scale two-stage AD plant, composed of a hydrolysis/acidogenesis (H) and an acetogenesis/methanogenesis (M) tanks, was monitored during feedstock substitution. Energy crop (triticale) was replaced by poultry manure litter and olive mill pomace. The increase percentage of poultry manure litter (up to 8.6%) and olive mill pomace (up to 30.5%) in the recipe incremented the total solids (up to 21% in H) and, consequently, the nitrogen content in the digestate (6.7 g N/kg in the solid fraction in H and 4-5 g NH4+-N/L in the liquid fraction). This favored the growth of Lactococcus sp. with consequent increment of lactate production (∼ 1 mg L-1 last two days of the survey) and the establishment of Weissella and Lactobacillus spp. Syntrophic acetate-oxidizers, including Syntrophaceticus (6% ± 1.7%), were detected manly in M but were negatively affected by the addition of the poultry manure litter, while the sulfate-reducing bacteria correlated with the variations of the volatile fatty acids. Planctomycetes putatively capable of anammox process were also found in the H during the first two days of the survey and accounted for 0.3 ± 0.01% of the total bacterial community. The stability of the process during feedstock change is the result of the shift of bacterial populations of different functional groups that showed peculiar adaptation patterns in the two stages of the plant.

Feasibility of removing surface deposits on stone using biological and chemical remediation methods.

The study was conducted on alterations found on stone artwork and integrates microbial control and a biotechnological method for the removal of undesirable chemical substances. The Demetra and Cronos sculptures are two of 12 stone statues decorating the courtyard of the Buonconsiglio Castle in Trento (Italy). An initial inspection of the statues revealed putative black crusts and highlighted the microbial contamination causing discoloration. In 2006, the Cultural Heritage Superintendence of Trento commissioned us to study and remove these chemical and biological stains. Stereomicroscopy characterised the stone of the sculptures as oolitic limestone, and infrared analyses confirmed the presence of black crusts. To remove the black crusts, we applied a remediation treatment of sulphate-reducing bacteria, which removes the chemical alteration but preserves the original stone and the patina noble. Using traditional and biomolecular methods, we studied the putative microbial contamination and confirmed the presence of biodeteriogens and chose biocide Biotin N for the removal of the agents causing the discolouration. Denaturing gradient gel electrophoresis fluorescent in situ hybridisation established that Cyanobacteria and green algae genera were responsible for the green staining whereas the black microbial contamination was due to dematiaceous fungi. After the biocide Biotin N treatment, we applied molecular methods and demonstrated that the Cyanobacteria, and most of the green algae and dematiaceous fungi, had been efficiently removed. The reported case study reveals that conservators can benefit from an integrated biotechnological approach aimed at the biocleaning of chemical alterations and the abatement of biodeteriogens.

Esterase as an enzymatic signature of Geodermatophilaceae adaptability to Sahara desert stones and monuments.

AIM: To assess esterase profiling of members of Geodermatophilaceae isolated from desert stones and monuments in Tunisia and Egypt. METHODS AND RESULTS: Members of Geodermatophilaceae family isolated from desert stones and monuments in Tunisia and Egypt were characterized by partial 16S rRNA sequences. Twenty-five strains were clustered in three dissimilar groups of the genera Geodermatophilus (12 strains), Blastococcus (5 strains) and Modestobacter (3 strains). Isolates were also screened and typed based on major groups of esterase hydrolytic activity. Their esterase patterns were determined and compared to those of ten reference strains belonging to Geodermatophilaceae family. Strains exhibited a diverse and complex pattern of electrophoretic esterase bands, and 31 haplotypes were obtained for the 35 investigated strains. Esterases produced by members of Geodermatophilaceae family have an optimal activity around 40 degrees C and at pH 8. Esterases from Geodermatophilus strains display a high resistance to thermal inactivation and alkaline pH and retaining 30 and 20% of activity after heating for 20 min at 120 degrees C and at pH 12, respectively, and were completely inactivated after 30 min at 120 degrees C. Enzyme activity has been strongly activated in the presence of Ca(2+)and Mg(2+) ions and moderately by Zn(2+) and was markedly inhibited by Cu(2+) and Co(2+) ions. CONCLUSIONS: Geodermatophilaceae isolates share a rich and particular pool of esterase activities that could be directly linked to harsh conditions characterizing their ecological habitat including high level of aridity, temperature, ionic strength and low nutrient availability. SIGNIFICANCE AND IMPACT OF THE STUDY: Esterase could be considered as enzymatic signature that outlines adaptability of Geodermatophilaceae in arid area.

Biochemical profiles, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA) for typing Staphylococcus aureus isolated from dairy products.

The study concerns 130 Staphylococcus aureus strains isolated from different raw-milk dairy products (122 isolates) and human samples (eight isolates). Four different typing techniques were applied: biochemical profiles (Biolog GP), restriction fragment length polymorphism of coagulase gene (coaRFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA). Moreover multiplex-PCR was used to study the distribution of genes encoding staphylococcal enterotoxins. The results of this study reveal marked genomic and phenotypic variability among the tested S. aureus. The considered techniques were all found useful for strain typing, but, based on discriminatory power as the key parameter of the typing system, MLVA and Biolog GP were found to be the most powerful techniques. The methods showed little concordance in terms of discerning the clusters of related strains.

Heteroduplex structures in 16S-23S rRNA intergenic transcribed spacer PCR products reveal ribosomal interoperonic polymorphisms within single Frankia strains.

AIMS: Detection of polymorphisms in intergenic transcribed spacer (ITS) 16S-23S rRNA within single Frankia strains. METHODS AND RESULTS: Polymorphisms in the 16S-23S rRNA ITS were investigated in single-colony subcultures of seven Frankia isolates. Multiple ITS-polymerase chain reaction (PCR) bands were detected solely in isolates BMG5.5 and BMG5.11. The slow-migrating bands in the ITS-PCR agarose gel electrophoresis profiles of the isolates were revealed to be heteroduplexes on the basis of their migration shift in different electrophoretic matrices, southern hybridization and the single-strand DNA mung bean endonuclease digestion. Laser-scanned capillary electrophoresis detected two ITS-PCR fragments differing in length by three and six nucleotide insertions/deletions in strains BMG5.5 and BMG5.11, respectively. Sequence analysis of the cloned ITS showed that in strain BMG5.5 the two ITS differed by the presence of three to four copies of the 3-bp tandem repeat 5'-TGG-3'. In strain BMG5.11, the two ITS differed by the presence of two to three copies of the 6-bp tandem repeat 5'-CTTGGG-3'. CONCLUSIONS: We demonstrate the occurrence of ITS 16S-23S rRNa polymorphisms within single Frankia strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We reported the occurrence of ITS 16S-23S rRNA polymorphisms within single Frankia strains from Elaeagnus host group recognized as the more flexible strains within Frankia genus. Furthermore, we underscored the applied interest of strains BMG5.11 and BMG5.5 in future ecological studies using ITS 16S-23S rRNA as molecular marker.

The autolytic phenotype of the Bacillus cereus group.

AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.

Biodiversity of Geodermatophilaceae isolated from altered stones and monuments in the Mediterranean basin.

An investigation was made into the occurrence and biodiversity of Geodermatophilaceae on 78 samples of altered stone surfaces from 24 monuments and natural stones in the Mediterranean basin; it was found that the total microbial counts ranged between 0 and 10(7) cfu g(-1) dry weight. Members of the Geodermatophilaceae family were isolated from 22 of the 78 samples examined, with the incidence of Geodermatophilaceae colonies in the cultivable population ranging from 1% to 100%. The highest percentage was found in six samples of markedly deteriorated stone. Sixty-five strains randomly isolated from the plates were clustered in six different groups by amplified 16S rDNA restriction analysis (ARDRA) using five different restriction enzymes. Twenty-five strains, representing all the ARDRA haplotypes, were characterized further by partial sequencing (350-550 bp) of the 16S rDNA and by analysing 76 morphological, metabolic and physiological properties. The strains were associated with three well-separated clusters of the genera Geodermatophilus, Blastococcus and Modestobacter. On the basis of 16S rDNA sequence and ARDRA analysis, only two strains were found to be related to the two reference strains of Geodermatophilus. All the others could be grouped with Blastococcus aggregatus (19 strains) or the Antarctic species Modestobacter multiseptatus (44 strains), suggesting that it is these two groups, rather than Geodermatophilus, that tend to colonize the stone surfaces, and that Modestobacter-like strains are also found in temperate/Mediterranean climates. From the BOX-polymerase chain reaction (PCR) data, it can be seen that the Modestobacter-like strains, belonging to the most represented ARDRA haplotype (haplotype B, 34 strains), are very polymorphic and that, over a stone surface, there is a wide genetic diversity at the microsite level.

Response of bacterial community during bioremediation of an oil-polluted soil.

AIM: To study the response of the bacterial community to bioremediation of a soil with an aged contamination of crude oil. METHODS AND RESULTS: The bacterial community in laboratory soil columns during a 72-day biostimulation treatment was followed by analysing the number of total cultivable hydrocarbon-degrading bacteria, soil respiratory activity and the 16S-23S rDNA internal transcribed spacer homoduplex heteroduplex polymorphisms (ITS-HHP) of total soil bacterial DNA. ITS-HHP permits an estimate of both length and sequence polymorphism in a 16S-23S rDNA spacer population, using to advantage the homoduplex and heteroduplex fragments that are generated during PCR. The treatment, made by air sparging and biostimulation with a mineral nutrient and surfactant solution, resulted in a 39.5% decrease of the total hydrocarbon content. Within 4 days of treatment onset the bacterial community underwent a first phase of activation that led to a substantial increase in the observable diversity. Subsequently, after a 12-day period of stability, another activation phase was observed with further shifts of the community structure and an increase in the abundance and diversity of catechol-2,3-dioxygenase (C23O) genes. CONCLUSIONS: The overall data suggest an important contribution of uncultivable bacteria to the soil bioremediation, since, during the second activation phase, the increases of the respiratory activity, bacterial diversity and C23O gene abundance and diversity were not accompanied by a corresponding increase of the cultivable bacteria number. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that successive phases of activation of bacterial populations occur during a bioremediation treatment of oil-polluted soil.

Genetic relationship in the 'Bacillus cereus group' by rep-PCR fingerprinting and sequencing of a Bacillus anthracis-specific rep-PCR fragment.

AIMS: To evaluate the genetic relationship in the Bacillus cereus group by rep-PCR fingerprinting. METHODS AND RESULTS: A collection of 112 strains of the six species of the B. cereus group was analysed by rep-PCR fingerprinting using the BOX-A1R primer. A relative genetic distinctness was found among the species. Cluster analysis of the rep-PCR patterns showed clusters of B. thuringiensis strains quite separate from those of B. cereus strains. The B. anthracis strains represented an independent lineage in a B. cereus cluster. The B. mycoides, B. pseudomycoides and B. weihenstephanensis strains were clustered into three groups at some distance from the other species. Comparison of sequences of AC-390, a typical B. anthracis rep-PCR fragment, from 27 strains of B. anthracis, B. cereus, B. thuringiensis and B. weihenstephanensis, representative of different clusters identified by rep-PCR fingerprinting, confirmed that B. anthracis diverges from its related species. CONCLUSIONS: The genetic relationship deduced from the rep-PCR patterns indicates a relatively clear separation of the six species, suggesting that they can indeed be considered as separate units. SIGNIFICANCE AND IMPACT OF THE STUDY: rep-PCR fingerprinting can make a contribution in the clarification of the genetic relationships between the species of the B. cereus group.