Microbial evolution during degradation of fungicides in an organic biomixture

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

[P-E.17] Direct cloning of heavy metal resistance genes from metagenomic DNA M. Golebiewski ∗ , E. Deja-Sikora, A. Tretyn Department of Biotechnology, Faculty of Biology and Earth Sciences, Nicolaus Copernicus University, Torun, Poland Keywords: metagenomics; heavy metal resistance; polluted soils Soil Prokaryotes are currently considered a pool of greatly variable and useful genes. Novel environmental genes isolated from this pool can be successfully used in industrial processes as well as for environment bioremediation. Microbes carry many resistance genes which enable their existence in conditions that are very harmful for other organisms. Unfortunately, only about 0.3% of soil microbial diversity can be recovered in pure cultures using standard microbiological media, leaving the majority still unknown. Until now, the only way to explore the genetic material of those “unculturable” bacteria is isolating total DNA (metagenomic DNA) from environmental samples. Purified metagenomic DNA can be subsequently used for environmental library construction. The average length of inserts is related to further libraries’ applications, which comprise their direct sequencing or screening. The most frequent screening strategy is based on testing clones on special media in order to find colonies displaying the function of interest.Objects of our research are prokaryotic heavy metal resistance genes that can be potentially applied for bioremediation of contaminated soils. We recovered environmental DNA from three samples coming from area adjacent to mine of zinc and lead ores, near Olkusz (southern Poland). Purified DNA fragments, with average length about 1520 kb, were enzymatically processed and then used for construction of three metagenomic libraries. For every library a collection of 40 000 clones was preserved. The clones were screened for specific function – resistance to cadmium, lead, copper and chromate. Functional screening was carried out on modified Tris-buffered solid media supplied with heavy metals. We have identified a few resistant clones which are now under investigation. To our knowledge this is the first study of direct cloning heavy metal resistance genes from metagenomic DNA.

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2004; Wu et al., 2008; Souza et al., 2009). However, it is known that several factors reduce the biodegradation rates in soil bioremediation process. Low solubility and adsorption on soil organic matrix are two major features that limit hydrocarbons availability in the soil water phase where microorganisms are active. Therefore the aim of the this study is to investigate the evolution of bacterial community during the removal of diesel from soil using laboratory scale bioreactors evaluating the influence of compost, surfactants (␤-Cyclodextrin) and bacteria consortium addition (previously selected from diesel-contaminated soil). The evolution of bacterial community during hydrocarbon removal was evaluated using both traditional culturable technique and a molecular culture-independent method such as Denaturing Gradient Gel Electrophoresis (DGGE). Petroleum hydrocarbons was determined in capillary gas chromatography (GC)/flame ionization detector (FID). Results of culturable heterotrophic bacteria population indicated that the compost addition positively influenced presence and development of total heterotrophic bacteria. DGGE analysis showed that biodiversity improved with the hydrocarbon removal and the restoration of biodiversity was positively influenced by the inoculum of bacteria consortium. Moreover, in presence of bacteria consortium was obtained the best hydrocarbon removal (96%) in comparison with the trials without bacteria consortium (87%) and control test (without compost and microbial consortium) (45%). References Antizar-Ladislao, B., Lopez-Real, J.M., Beck, A.J., 2004. Bioremediation of polycyclic aromatic hydrocarbon (PAH) – contaminated waste using composting approaches. Crit. Rev. Environ. Sci. Technol. 34, 249–289. Souza, D.A., Chinalia, F.A., Foresti, E., Zaiat, M., 2009. Bioremediation of gasolinecontaminated groundwater in a pilot-scale packed-bed anaerobic reactor. International Biodeterioration & Biodegradation 63, 747–751. Wolicka, D., Suszek, A., Borkowski, A., Bielecka, A., 2009. Application of aerobic microorganisms in bioremediation in situ of soil contaminated by petroleum products. Bioresource Technology 100, 3221–3227. Wu, Y., Luo, Y., Zou, D., Ni, J., Liu, W., Teng, Y., Li, Z., 2008. Bioremediation of polycyclic aromatic hydrocarbons contaminated soil with Monilinia sp.: degradation and microbial community analysis. Biodegradation 19, 247–257.

doi:10.1016/j.jbiotec.2010.09.040 [P-E.19]

doi:10.1016/j.jbiotec.2010.09.039 [P-E.18] Monitoring of bacterial community during a bioremediation process of diesel-contaminated soil Manuela Taccari 1,∗ , Vesna Milanovic 1 , Francesca Comitini 1 , Cristiano Casucci 2 , Maurizio Ciani 1 1

Università Politecnica delle Marche, Dipartimento SAIFET, Italy Università Politecnica delle Marche, Dipartimento SAPROV, Italy Keywords: Bioremediation; Diesel-contaminated soil; Bacterial community; DGGE

Microbial evolution during degradation of fungicides in an organic biomixture Vesna Milanovic 1,∗ , Laura Coppola 2 , Francesca Comitini 1 , Cristiano Casucci 2 , Costantino Vischetti 2 , Maurizio Ciani 1 1

Università Politecnica delle Marche dipartimento SAIFET, Italy Università Politecnica delle Marche, dipartimento SAPROV, Italy Keywords: Fungicides; Biobeds; Biodegradation; Microbial biomass; DGGE 2

2

Petroleum hydrocarbons are widespread environmental contaminants and hotspots pollution, as a result of spills, leaks and improper disposal are common source of public concern and health and safety risk assessments. Among hydrocarbon pollutants, diesel is a complex mixture of alkanes and aromatic compounds that frequently are reported as soil contaminants (Wolicka et al., 2009). Bioremediation has been considered as one of the potential methods for the cleanup of petroleum-hydrocarbon contaminated sites. Indeed, the microbial ability to degrade these aromatic compounds is the success of this advance (Antizar-Ladislao et al.,

Biological systems are being developed all over the EU countries to protect water-bodies from pesticides contamination at farm level. Biobeds are on-farm biological system developed in Sweden to retain and attenuate pesticides contamination coming from inappropriate procedures at pesticide mixing and handling sites and inappropriate disposal of pesticide sprayer rinse water (Coppola et al., 2007; Castillo et al., 2008). The experiment was conducted when equipments were washed in a biobed after treatments. Commercial formulates of dimetomorph(DF), penconazole(PC), azoxystrobin(AZ), metalaxyl(MX), fludioxonil(FL) and cyprodinil(CY) were mixed and downloaded onto the bio mixture following concentrations and time schedule of treatments for grapevine season during 112 days. Pesticides degradation was monitored by measuring the residual concentration over the time.

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Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

The evolution of microbial community were monitored using culture-dependent and independent methods such as viable plate count and Denaturing Gradient Gel Electrophoresis (DGGE) to describe the influence of fungicides on microbial diversity. Relation between microorganisms variation and fungicide degradation was investigated to improve the system efficiency. Results showed a good capability of biomix to degrade pesticides. At the end of the experimentation time the concentration of most of pesticides was nearly to the complete degradation. No significant differences were observed in culture-dependent heterotrophic bacterial and fungal evolution during the treatment. DGGE analysis of bacterial community after two consecutive treatments with PC+DF and after the treatment with AZ+CY+FL showed the highest differences between treated samples and their controls. DGGE analysis showed a strong reduction of fungal community after PC+DF addition (10th and 23th day). At 64th day fungicides were almost degraded and an evident enhancement of fungal biodiversity with the appearance of new species was shown. Interestingly, at the end of the experiment the microbial community exhibited a strong similarity between treated and untreated sample indicating that the variations of microbial community was only temporally.

References Castillo, M., Tortensson, d.P., Stenstrom, L.J., 2008. Biobeds for environmental protection from pesticide uses. A review. J. Agric. Food chem 56 (15), 6206–6219. Coppola, L., Castillo, M., Monaci, d.P., Vischetti, E.C., 2007. Adaptation of the biobed composition for chlorpyrifos degradation to southern Europe conditions. J. Agric. Food Chem. 55, 396–401.

doi:10.1016/j.jbiotec.2010.09.041 [P-E.20] Negative chemotaxis to trichloroethylene and its chemosensory protein in Pseudomonas putida F1 S. Oku ∗ , M. Hayashida, T. Tajima, Y. Nakashimada, J. Kato Hiroshima University, Japan Keywords: Chemotaxis; Biodegradation; Trichloroethylene; Pseudomonas putida F1 Pseudomonas putida F1 is a toluene-oxidizing bacterium, which is capable of degrading trichloroethylene (TCE), an important environmental pollutant, in the presence of toluene. This bacterium has been extensively studied as a bioremediation agent in TCE-polluted environments. Parales et al. reported that P. putida grown in the presence of toluene showed positive chemotactic responses toward TCE (Appl. Environ. Microbiol. 66, 4098, 2000). Although P. putida F1 were attracted to agarose plugs containing high concentrations of TCE, they never touched the plugs. We speculate that this phenomenon is attributed to negative chemotactic responses to TCE. The migration of TCE-degrading bacteria toward TCE might speed up the biodegradation process because it should bring the cells into contact with TCE. Conversely, negative chemotaxis to pollutants might have negative effects on biodegradation process. In this study, we investigated negative chemotaxis to TCE in P. putida F1. Computer-assisted capillary assays revealed that P. putida F1 grown in the absence of TCE were repelled by TCE. In the previous study, we demonstrated that PctA, PctB, and PctC, chemosensory proteins for amino acids, was involved in negative chemotaxis to TCE in Pseudomonas aeruginosa PAO1. Blast search found a P. putida F1 gene showing a high similarity to P. aeruginosa pctA (designated pctAPp ). Complementation analysis using P. aeruginosa
pctABC and pctAPp demonstrated that PctAPp also recognized amino acids as attrac-

tants. We inactivated the genomic pctAPp gene in P. putida F1. The resulting mutant was attracted to low concentrations of TCE even though it was grown in the absence of toluene. The mutant grown in the presence of toluene showed positive responses to higher concentrations of TCE to which the wild type did not respond. These results clearly demonstrate that P. putida F1 is repelled by TCE under certain conditions and PctAPp is involved in the negative chemotaxis to TCE. doi:10.1016/j.jbiotec.2010.09.042 [P-E.21] Biotreatment of gas-phase VOC mixtures from fibreglass and composite manufacturing industry E. Rene ∗ , M. Montes, M.C. Veiga, C. Kennes University of La Coru˜ na, Spain Keywords: Acetone; toluene; styrene; biofilter (BF); Sporothrix variecibatus; elimination capacity (EC); removal efficiency (%) Introduction: Acetone, toluene and styrene (ATS), representative air-pollutants arising from the use of resins, solvents, paints, thinners, foaming agents, and gel-coats during the production process in fibreglass and composite manufacturing industries cause significant environmental nuisance. The health hazards associated with these emissions and increased attention from the regulatory authorities have helped such industries to adopt appropriate pollution control technologies. Fungal BFs are beneficial, versatile and efficient for handling large volumes of VOCs at moderately high loading rates (Kennes et al., 2009). Methods: Experimental: A previously isolated yeast-like fungus Sporothrix variecibatus (Fig. 1) was inoculated in a perlite BF having a bed-volume of 0.0019m3 . Fresh mineral salt medium was added periodically from the top, once every 3d (Rene et al., 2009). Experiments were conducted at EBRTs of 68.4, 34.2 and 17.1 s, and by varying the concentrations of ATS from 0.03-3.27, 0.05-2.3 and 0.08-3.9 gm-3 , respectively.Analytical: Styrene concentration in gas samples was measured by gas chromatography on a HP-5890 GC, using a 50m TRACER column and a FID for detection.

Fig. 1. SEM photograph of the original Sporothrix sp. Results and Discussion: Styrene was better removed (47-100%) in the BF in comparison to both acetone (34-100%) and toluene (42100%). In the concentration range tested, the maximum EC achieved were 108, 72 and 144 gm-3 h-1 , for ATS, respectively. Besides, it was observed that, increasing the concentration of ATS, tends to