Annals of Microbiology

Information gleaned from soil microbiota may provide access to new economically important species. Here, we describe the isolation, identification, and genetic diversity of high-density bacterial populations isolated from aged oil-contaminated soil. Twenty different morphotypes were identified in populations present at densities of up to 107 cells g−1 soil, encompassing seven bacterial genera based on 16S rRNA sequencing. Six isolates of the genus Bacillus were identified, three of which appear to consume oil. The genetic clusters defined by the DNA fingerprinting analysis suggest that there is a close relationship between these oil-eating Bacillus species. Isolates able to grow using crude oil as a carbon source were biochemically characterized and found to exhibit high lipolytic activity in liquid medium and to produce alkaline-stable biosurfactants. Fluorescence spectroscopy analysis of the cell-free extract from the oil-eating Bacillus sp. strain MO.04b showed an increase in the relative fluorescence intensity of low-molecular-mass aromatics concomitantly with an increase in the protein content, suggesting the transformation of aromatic hydrocarbons to the liquid phase in response to biodegradation. The approach adopted in this study suggests a low diversity of the high-density bacterial population colonizing an aged oil-contaminated soil and may prove useful in selecting bacterial isolates for bioremediation studies and biotechnological applications such as biosurfactant production.

Strain SFA13, with the ability to perform simultaneous heterotrophic nitrification and aerobic denitrification, was isolated from the Songhua River. Investigation of the phenotypic and genotypic characteristics of strain SFA13 revealed that its 16S rRNA gene sequence shares high similarity with that of Microbacterium esteraromaticum DSM 8609T (99.78 %). gyrB and recA gene sequences showed 92.40 % and 92.57 % similarity to those of M. esteraromaticum DSM 8609T, respectively, showing that strain SFA13 is a member of M. esteraromaticum. However, strain SFA13 showed distinctly different phenotypic characteristics from the type strain DSM 8609T. Strain SFA13 was negative for H2S production, β-fucosidase, hydrolysis of starch and Tween, utilization of salicin, l-alanine, d-fructose, d-galactose, d-glucose and d-trehalose; and positive for alkaline phosphatase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and β-galactosidase. The ammonium removal rate of strain SFA13 was affected by temperature, decreasing obviously as the temperature dropped from 30 °C to 10 °C. Even so, strain SFA13 exhibited a high ammonium removal efficiency at 5 °C. The highest ammonium removal rate was 4.41 mg NH4 + L−1 h−1 when the initial ammonium concentration was 61.63 mg L−1 at 5 °C. Excessive ammonium (more than 62 mg L−1) restricted the ammonium removal rate of strain SFA13. This strain could be used effectively to remove low concentrations of ammonium ion at low temperature.

Waste rubber has posed challenging environmental and disposal problems across the world. This study focused on the microbial reclaiming of ground tyre rubber (GTR) by Acidithiobacillus ferrooxidans YT-1 cultured in media with variable Fe2+ concentrations. The Acidithiobacillus ferrooxidans YT-1 strain with the ability of oxidizing sulfur and reclaiming waste rubber was isolated and identified. Toxicity tests of different rubber and additives in tyre rubber compounds to microorganisms was quantitatively investigated. After desulfurization, there were many small colonies on the surface of the desulfurizated GTR (DGTR), due to surface degradation by A. ferrooxidans YT-1. The amount of small colonies increased and sulfur content decreased with the increase of Fe2+ concentrations in the media, implying that Fe2+ concentration had a great influence on the degradation ability of A. ferrooxidans YT-1. A medium with a high Fe2+ concentration was good for growth of A. ferrooxidans YT-1. Compared with styrene butadiene rubber (SBR)/GTR blends, the tensile strength and elongation at the break of the SBR/DGTR blends were significantly improved. The scanning electron microscope (SEM) photographs of the fracture surface further indicated a good coherency between DGTR and the SBR matrix. These results revealed that A. ferrooxidans YT-1 cultured in a medium with a high Fe2+ concentration could improve the reclaiming efficiency of waste rubber.

The aim of the present study was to investigate the tolerance of five new Achromobacter and Pseudomonas strains to kerosene and to establish if the production of several secondary metabolites increases or not when these bacteria were grown in the presence of kerosene. The biodegradation of kerosene by isolated bacteria was also investigated in this study. Five Proteobacteria were isolated from different samples polluted with petroleum and petroleum products. Based on their morphological, biochemical, and molecular characteristics, isolated bacteria were identified as Achromobacter spanius IBBPo18 and IBBPo21, Pseudomonas putida IBBPo19, and Pseudomonas aeruginosa IBBPo20 and IBBPo22. All these bacteria were able to tolerate and degrade kerosene. Higher tolerance to kerosene and degradation rates were observed for P. aeruginosa IBBPo20 and IBBPo22, compared with that observed for A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19. All these bacteria were able to produce several secondary metabolites, such as surfactants and pigments. Glycolipid surfactants produced by P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 have a very good emulsification activity, and their activity increased when they were grown in the presence of kerosene. The production of rhamnolipid surfactants by P. aeruginosa IBBPo20 and IBBPo22 was confirmed by detection of rhlAB gene involved in their biosynthesis. Pyocyanin and pyoverdin pigments were produced only by P. aeruginosa IBBPo20 and IBBPo22, while carotenoid pigments were produced by all the isolated bacteria. Significant changes in pigments production were observed when P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 were grown in the presence of kerosene. Due to their ability to tolerate and degrade kerosene, and also to produce several secondary metabolites, the isolated bacteria could be used in the bioremediation of kerosene-polluted environments.

Prodigiosin has attracted great interest for several decades for its proapoptotic anticancer and other activities. However, the low productivity of prodigiosin under submerged fermentation (SmF) limits its commercial application. In this study, S. marcescens Xd-1 was newly isolated from a mouldy tofu sample and was used to produce prodigiosin by solid-state fermentation (SSF) using bagasse as an inertia matrix. The result showed that S. marcescens Xd-1 was adapted to SSF for prodigiosin production and could bear a higher concentration of glycerol. Process parameters were systematically examined to improve the conversion of glycerol to prodigiosin by a response surface methodology. The maximal yield of prodigiosin (40.86 g kg−1 dry solid) was achieved with glycerol 1.17 g g−1 bagasse, soy peptone 0.33 g g−1 bagasse, an initial moisture content of 83.5 %, and 1-mm particles of bagasse by using a response surface methodology (RSM). In addition, the extraction ratio of prodigiosin increased rapidly to 90 % in 30 min by using ultrasonic-assisted reflux extraction. Our results expand the culture method of S. marcescens for prodigiosin production and enhanced the conversion of glycerol to prodigiosin.

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) fromTrichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme fromSaccharomycopsis fibuligera significantly hindered its expression and secretion inSaccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes inS. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) inS. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenisedS. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain ofS. cerevisie, WM91-CBGL1, which exhibited up to 200 U L−1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with theT. resei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.

The aptitude of twentySaccharomyces sensu stricto strains to remove ochratoxin A from a synthetic medium containing 1.1 ng/mL, about half of the European Community limit, was evaluated using four to six mg of biomass (wet weight)/mL. Seven satins show high levels of ochratoxin A removal, 0.72-1.10 ng/mL, equivalent to 66–100% of the available toxin, and unitary removing ativity of 14.31–27.24 pg/mg of biomass. Further research will be carried out to study the mechanism of OTA removal and to confirm the ability of the most efficacious strains ofSaccharomyces sensu stricto to remove OTA from contaminated wort and grape must during alcoholic fermentation.

Catechol 1, 2-dioxygenase (1, 2-CTD) plays an important role in the biodegradation of most aromatic pollutants (e.g. phenol) by cleaving the bond between the phenolic hydroxyl groups. In this study, we analysed microbial populations in petroleum-contaminated soil samples by the culture-dependent approach and chose one population as a sample location for metagenomic studies. This population showed the largest number of phenol-degrading isolates and the most phylogenetic diversity based on repetitive sequence-based PCR and 16S rDNA analyses. The isolates were assigned mainly to the bacterial genera Acinetobacter and Pseudomonas. To enable maximum recovery of 1, 2-CTD coding sequence from the genomic pool of bacterial populations, we extracted the substrate-induced RNA, reverse-transcribed it to the specific cDNA using primers designed for this purpose and constructed a metagenomic library. Alignment analysis of one recombinant sequence indicated a significant divergence, with the 1, 2-CTD gene of Pseudomonas aeruginosa DK2 as the closest species according to sequence similarity. This result indicates that such specifically designed primers are likely suitable candidates as biomarkers to construct 1, 2-CTD gene-targeted metagenomic libraries. By sequencing the larger clones of this metagenomic library, we hope to obtain novel and unpredicted 1, 2-CTD genes that can contribute to our knowledge of microbial diversity of these genes.

The main objective of this work was to develop a protocol for the detection and quantification of ochratoxin A (OTA) to be used directly in food commodities, where sensitivity and robustness are critical factors. In this study, the contamination of wine grapes (var. Merlot) by OTA producer Aspergillus carbonarius was detected by PCR on asymptomatic grape berries. Four varieties of Vitis vinifera harvested directly from the field were used in the PCR-based assay. In the experiments, A. carbonarius was used to develop the PCR method, and Aspergillus niger strain 7096 and other species isolated from grapes were also used to test the specificity of the method presented below. The multiplex PCR reaction was tested on fungal DNA by amplifying two target genes in order to differentiate with an higher degree of effectiveness potentially ochratoxigenic black Aspergilli from other fungal contaminants. These PCR-based assays showed high sensitivity and effectiveness in detecting A. carbonarius from grape DNA. The same grape berries in which A. carbonarius DNA was revealed at an early timepoint showed the visible presence of black rot after appropriate incubation, confirming the results obtained by the molecular methods developed here. Furthermore, OTA detection on Merlot wine was performed with a novel device based on the use of an amorphous silica photosensor. This technique, using very simplified and rapid extraction procedures, allowed the detection and quantification of OTA from wine contaminated at 2 ppb. These methods allow rapid and early detection of the presence of the pathogen and of the toxin within a working day.