environmental biotechnology lectures l15 - actabp.pl · environmental biotechnology lectures ......

Environmental biotechnology

Lectures

L15.1

Biodegradation of biodiesel/diesel blends in soils: effects on hydrocarbon dissipation and natural microbial communitiesŁukasz Chrzanowski

Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznań, Polande-mail: Łukasz Chrzanowski <[email protected]>

It is generally considered that the addition of biodiesel has a positive effect on biodegradation of diesel fuel hydro-carbons. Although the stimulating effect has been widely recognized, the exact mechanisms behind this phenom-enon are still not entirely elucidated. While the presence of biodiesel may induce dispersion of diesel fuel hydrocar-bons, thereby increasing their overall bioavailability to the degrading microorganisms, its effect on the biodegradation of specific hydrocarbon fractions may differ. This may di-rectly correspond to quantitative and qualitative changes among the microbial community of hydrocarbon degrad-ers involved in the biodegradation processes. In order to investigate this issue we studied the dissipation of specific hydrocarbon fractions during the biodegradation of die-sel/biodiesel blends carried out by soil-isolated microbial consortia under different aeration regimes. The community dynamics was assessed by employing real-time PCR and the ddCt method for relative quantification. Interestingly, the obtained results suggested, that FAMEs constituting biodiesel were degraded at the same rate as diesel deriving alkanes. This led to differences in the biodegradation of toxic aromatic fuel constituents and contribute to changes among the growth rates of specific degraders. The evalua-tion of biodiesel effect was carried out in laboratory condi-tions as well as during field studies.

L15.2

Yeast metabolic engineering for production of fuels and chemicalsAndriy A. Sibirny1,2

1Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine; 2University of Rzeszow, Polande-mail: Andriy Sibirny <[email protected]>

This lecture summarizes author’s own and literature data on metabolic engineering of non-conventional yeasts Hansenula polymorpha and Pichia stipitis for improvement of ethanol production from lignocellulosic sugars, mainly from most abundant pentose sugar xylose. H. polymorpha is the thermotolerant yeast capable of alcoholic fermenta-tion of glucose, cellobiose and xylose at high temperatures (48ºC), however, ethanol yields from xylose in wild-type strains was very low. Construction of the advanced strains with enhanced ethanol production from xylose included several steps of classic mutagenesis and selection as well as experiments using approaches of metabolic engineering. Increase in ethanol production was achieved by introduc-tion of mutation 2Eth — leading to defect in ethanol uti-lization. Further increase in xylose alcoholic fermentation were obtained by overexpression of bacterial xylA gene coding for xylose isomerase and own genes XYL2 and XYL3 coding for xylitol dehydrogenase and xylulokinase, respectively. Alternatively, increase in ethanol yield from xylose was achieved by overexpression of engineered xy-lose reductase (gene XYL1) together with native XYL2 and XYL3. Additional increase in ethanol yield was obtained after overexpression of PDC1 coding for pyruvate decar-boxylase. In P. stipitis, overexpression of engineered xylose reductase and native xylitol dehydrogenase and xyluloki-nase also led to increase in ethanol yield from xylose.Second part of this presentation describes experiments of the author’s team on metabolic engineering of flavinogenic yeast Candida famata for construction of the robust produc-er of riboflavin (vitamin B2) and flavin nucleotides FMN and FAD achieved due to identification of the novel tran-scription factor SEF1 as well as overexpression of genes SEF1, IMH3 (coding for IMP dehydrogenase), RIB1 (GTP cyclohydrolase II), RIB7 (riboflavin kinase), FMN1 (ribo-flavin kinase) and FAD1 (FAD synthetase).

Eurobiotech 2011 111

L15.3

Soil and water efficiency enhancing phytotechnologies (SWEEP)M. N. V. Prasad

Department of Plant Sciences, University of Hyderabad, Hyderabad, Indiae-mail: Prasad MNV <[email protected]>

Environmental pollution with heavy metals currently re-quire considerable attention. Plant based technologies (=Phytotechnologies) are considered to be SWEET (Soil and Water Efficiency Enhancing Technologies) for reduc-ing the burden of heavy metal load. Increased population, industrialization and urbanization are responsible for heavy metal load in the environment. Regulatory obligations to remediate such materials are cost prohibitive. ‘Dig and dump’ are invasive and destructive technologies result-ing in a loss of structure and function of the ecosystem. Therefore, plant-based technologies are favored being potentially sustainable, act as sinks for atmospheric car-bon and relatively low cost. They are especially appeal-ing for treatment or reuse of slightly or moderately con-taminated natural resources (water, soil and air). Advances in plant based science and technologies have enabled us to apply the potential of biological diversity for pollution abatement. This is emerging as an effective innovative tech-nology for treatment of a wide variety of contaminants. This technology includes plant and microbe interactions. Rhizoremediation, which is the most evolved process of bioremediation, involves the removal of specific contami-nants from contaminated sites by mutual interaction of plant roots and suitable microbial flora. Bioremediation is an invaluable tool box for wider application in the realm of environmental protection. Bioremediation approach is cur-rently applied to contain contaminants in soil, groundwater, surface water, or sediments including air. These technolo-gies have become attractive alternatives to conventional cleanup technologies due to relatively low capital costs and the inherently aesthetic nature. Mining industries, including abandoned mines release a variety of heavy metals.Large number of plants (natural and transgenic) and/or associ-ated with rhizosphere micro-organisms are extraordinarily active in these biological interventions and cleaning up pol-lutants by removing or immobilizing them. While diverse microbes are the most active agents, fungi and their strong oxidative enzymes are key players in degrading/ decontam-ination recalcitrant polymers and xenobiotic chemicals as well. Constructed wetlands are the result of human skill and technology since ages integrating the geology, hydrol-ogy and biology. Remediation–Eenergy–Health and Sus-tainability are discussed with appropriate examples.

Posters

P15.1

Proteome analysis of moderately halophilic bacteria response to oxygen limitationSelim Ceylan, Berna S. Akbulut, Dilek Kazan

Marmara University Engineering Faculty Bioengineering Department, Istanbul, Turkeye-mail: Selim Ceylan <[email protected]>

The moderately halophilic bacterium Halomonas sp. strain AAD12 can encounter microaerobic conditions in in-dustrial processes such as waste treatment. In industrial bioreactors, oxygen transfer and dispersion throughout the medium is often inconvenient, leading to regions of oxygen limitation. Bacteria under these regions will sense and respond to oxygen starvation, leading to switching to anaerobic metabolism. To examine oxygen limitation response of strain ADD12, cells were grown on aerobic and semi-aerobic conditions. Growth rates and cell yields were decreased by oxygen limitation. For both conditions two dimensional gel elec-trophoresis were conducted and proteome maps were visu-alized. The differently expressed proteins were analyzed by Proteome-Analyzer 4700. Proteins involved in oxygen limitation response were involved in transcription, energy metabolism, fatty acid and protein biosynthesis. The results showed that strain AAD12 could adapt to low oxygen conditions by modulating energy metabolism. Slower growth under low oxygen might be related with im-pairment of DNA metabolism. Flexible respiration signifi-cantly advantage on multiplication and long-term survival of halophilic strain AAD12 at stress conditions in indus-trial processes.

112 Eurobiotech 2011

P15.2

Bacterial changeability in aerobic — anoxic membrane bioreactors treating coke wastewaterAleksandra Ziembińska, Agata Karło, Grzegorz Cema, Sebastian Żabczyński

The Silesian University of Technology, Environmental Biotechnology Department, Gliwice, Polande-mail: Aleksandra Ziembinska <[email protected]>

One of the main energetic sources for industrial and com-munal purposes in Poland is coke, the solid residue of impure carbon obtained in distillation process after vola-tile material removal. During coke distillation, performed in coke-plants, a large amount of wastewater is released. These sewage contains toxic and harmful chemicals, such as: cyanides, thiocyanates and phenols. These substances are dangerous for water environment and they have de-structive influence on activated sludge microorganisms used for biological wastewater treatment. In wastewater treatment the nitrification is crucial for ammonia nitrogen removal, but nitrifiers, a group of microorganisms respon-sible for this process, are highly sensitive towards chemical contaminants. It is important to find an alternative way for nitrogen removal, that should be introduced in coke-waste-water plants as an effective and relatively cheap process. Anammox (Anaerobic Ammonium Oxidation) is regarded to be useful in such situation. Due to the fact, that Anam-mox bacteria are difficult to analyze using standard bacte-riological procedure, molecular biology techniques should be used. DGGE (Denaturing Gradient Gel Electrophoresis) is known to be the most suitable for microbial complex-ity studies, as a method enabling bacterial changeability and diversity analysis. The aim of this study was bacterial changeability monitoring in aerobic — anoxic membrane bioreactor system. Bioreactors were inoculated with acti-vated sludge from coke wastewater treatment plant and a synthetic coke wastewater was used as a medium. In the first aerobic membrane bioreactor, the phenol removal, or-ganic matter removal and the first step of nitrification took place. The followed anoxic bioreactor was aimed for the Anammox process. Due to the differences in aeration dur-ing the experiment in both bioreactors the diversification of bacterial community in inoculum was observed from the 3rd week of the experiment. Two groups of bacterial genotypes, varied with GC content, were discovered in the systems. The identification of dominant genotypes based on DNA sequencing will be performed.

P15.3

Determination of Monod kinetic parameters for wastewater containing imidazolium ionic liquids treated with activated sludge systemDorota Gendaszewska1, Ewa Liwarska-Bizukojc2

1Technical University of Lodz, Institute of Fermentation Technology and Microbiology, Lodz, Poland; 2Technical University of Lodz, Institute of Fermentation Technology and Microbiology, Lodz, Polande-mail: Dorota Gendaszewska <[email protected]>

Ionic liquids (ILs) are salts which occur in the liquid state over a wide temperature range. Wide use of these com-pounds in the industry increases the possibility of release of ILs into aquatic environment. They may become per-sistent pollutants in wastewater and as a consequence lead to surface water pollution. Due to their physicochemical properties ILs can influence on biological wastewater treat-ment including metabolism of activated sludge microor-ganisms. The aim of the study is to determine the values of kinetic parameters, i.e. maximum specific growth rate of heterotrophic biomass (µmax) and half saturation constant (Ks), for the processes of biological treatment of wastewa-ter containing imidazolium ionic liquids. Biological waste-water treatment was made with the use of activated sludge biomass. Both kinetic parameters were estimated on the basis of results obtained from oxygen uptake rate (OUR) tests. The tests were carried out in a laboratory scale batch reactor of working volume equal to 4 l and at constant temperature of 20ºC. Tests were conducted separately for each of three imidazolium ionic liquids studied, i.e. 1-ethyl-3-methylimidazolium bromide (IL1), 1-hexyl-3-methylimi-dazolium bromide (IL2) and 1-decyl-3-methylimidazolium bromide (IL3). Each ionic liquid was dissolved in the syn-thetic municipal wastewater at concentration of 50 mg/l. In addition, the control tests without ionic liquids were conducted. All tests were performed in triplicate. The val-ues of maximum specific growth rate of heterotrophic bio-mass exposed to wastewater containing ionic liquids were 0.23, 0.44, 0.65 h-–1 for IL1, IL2, IL3, respectively. This indicates that maximum specific growth rate of microor-ganisms increases with the elongation of alkyl chain length in the ILs cation. It occurred that the estimated values of µmax are higher than the values determined for wastewater without the addition of ionic liquids. The values of half saturation constant were 6.86, 7.25, 16.06 mg O2/l for IL1, IL2, IL3 respectively. It means that the affinity of substrate to biomass decreases with the increase in chain length of the alkyl substituent. AcknowledgementsThis work was financed by Ministry of Science and Higher Education (Republic of Poland), project no. N N209 102 637.


P15.4

Changes in modified plastics as a result of hydrolysis and biodegradation with filamentous fungiBożena Nowak, Jolanta Pająk

University of Silesia, Faculty of Biology and Environmental Protection, Department of Biochemistry, Polande-mail: Bożena Nowak <[email protected]>

The widespread use of plastics, especially for the produc-tion of short-life disposable products has resulted in their extensive accumulation in the environment. Plastics, espe-cially in the case of polystyrene or polyethylene, are ma-terials resistant to different environmental factors such as radiation, temperature, humidity and microorganisms. Re-sistance to degradation is due to their backbone solely built of carbon atoms. Contrary to polyethylene other polymers containing heteroatoms in the main chain e.g. polyesters are potentially susceptible to hydrolytic, abiotic or biotic, cleav-age. Additionally, it is generally accepted that aliphatic poly-esters are more easily degraded than aromatic polyesters. For practical applications often compositions or copoly-mers of different types are created so as to obtain desirable utility properties of plastics. Possible combinations of mix-ing various polymers results in difficulties of predicting the mechanism of their degradation. In this work the degree of hydrolytic and microbial degrada-tion of low-density polyethylene (LDPE) and poly(ethylene terephthalate) (PET) both modified with aliphatic polyes-ter Bionolle® was evaluated. After the experiments film weight loss was estimated and scanning electron micros-copy (SEM) analysis allowing estimation of colonization of plastics by microorganisms and observation of the types of changes on the material surface was performed. Important chemical changes in the polymeric chains were detected by Fourier Transform Infrared Spectroscopy (FTIR). The examination of changes in polymers showed how the ex-tracellular enzymes secreted by microorganisms attack the polymer chains. As a result of hydrolytic degradation it turned out that the degree of hydrolytic degradation was dependent on the content of aliphatic polyester in polymeric films. However, polyethylene films exhibited quite distinct behaviour when compared to poly(ethylene terephthalate) films. Increasing Bionolle® content in films accompanied increasing weight loss of polyethylene, exclusively. Biodegradation of both pre-treated compositions also proceeded in a different way. While, as during the hydrolysis, the degree of biodegrada-tion of modified polyethylene film increased with the con-tent of polyester Bionolle®, the degree of biodegradation of poly(ethylene terephthalate) films depended only on the fungal species used.

P15.5

Changes in bacterial fatty acid composition during phenol degradation in soilŻaneta Swędzioł, Agnieszka Mrozik

Department of Biochemistry, University of Silesia, Polande-mail: Swędzioł Żaneta <[email protected]>

Phenol contaminated soils are of specific environmental concern because of the ecological risk associated with its high toxicity and relatively high mobility in soil. The most efficient and cost effective method for removal of phenol from contaminated soils is biodegradation by microorgan-isms.The aim of this study was to test if fatty acid methyl ester (FAMEs) analysis could be used to monitor the progress of phenol removal from soil. The biodegradation studies were conducted in two independent experimental arrangements. The first comprised two sterile soils inoculated by single wild strain of Pseudomonas vesicularis and genetically modi-fied P. vesicularis (pBR322) and the second included non-sterile soils bioaugmented with P. vesicularis (pBR322) only.It was found that both strains after addition to sterile loamy soil (S1) and silty sand soil (S2) degraded phenol at the con-centration of 1.7 mg g–1 soil within 20 and 28 days, re-spectively. Simultaneously, no differences between catechol dioxygenases activity and survival of inoculants in these soils were observed. Soil bioaugmentation with P. vesicularis (pBR322) strain significantly enhanced soil activity towards phenol degradation in comparison with sterile soils. Phe-nol was completely removed from bioaugmented S1 and S2 soils within 8 and 12 days, respectively. It was also con-firmed that P. vesicularis (pBR332) and heterotrophic bacte-ria survived better in S1 soil containing 16.5% of organic mater than in L2 soil with small content of organic matter (1.9%). On day 8 in phenol-polluted S1 soil about 84% of inoculated cells and 79% of initial number of heterotroph-ic bacteria were determined, whereas in L2 soil on day 12 about 66% and 75%, respectively.Under phenol exposure significant alterations in FAMEs profiles of both strains inoculated into S1 and S2 soils were connected with the content of branched and cyclopropane fatty acids. The percentages of branched fatty acids and cyclopropane fatty acid 19:0cy ω8c increased significantly when 76–81% of the initial dose of phenol in sterile and bioaugmented soils was removed. Simultaneously, in that time the decline in 17:0cy amount was detected. Our stud-ies confirmed that FAMEs analysis was suitable for moni-toring the progress of phenol removal from contaminated soils. Moreover, some fatty acids were sensitive probes for degree of phenol utilization.P. vesicularis (pBR322) was the host of pBR322 plasmid which allowed to detect inoculated cells among indigenous microflora.


P15.6

Metabolic and symbiotic properties of genetically differentiated Rhizobium leguminosarum bv. trifolii strains derived from single soil environmentGrażyna T. Stasiak1, Andrzej Mazur1, Jerzy Wielbo1, Agnieszka Kubik-Komar2, Anna Skorupska1 1Maria Curie-Skłodowska University in Lublin, Department of Genetics and Microbiology, Lublin, Poland; 2Lublin University of Life Sciences, Chair of Applied Mathematics and Informatics, Lublin, Poland e-mail: Grazyna <[email protected]>

Bacteria have evolved a great diversity of metabolic strat-egies to survive in harsh environment such as soil and plant rhizosphere in which distribution of nutrition var-ies over time and space. Soil bacteria named rhizobia cre-ating symbiosis with legumes have large and multipartite genomes, consisting of chromosome and usually large plasmids. The size of the genome is influenced by envi-ronmental conditions. Rhizobia must survive and compete each other in a rhizosphere, where nutrients and oxygen are limited. Moreover bacteria may be faced with osmotic stress, drought and temperature changes. Genetic content of their large and dynamic genomes better adapt them to soil conditions. Our previous studies of Rhizobium legumi-nosarum bv. trifolii (Rlt) isolates from root nodules of clover plants growing in each other vicinity showed substantial divergence of their genome organization especially taking into account the content of plasmid DNA. The purpose of this study is to find correlation between the genomes di-versity of the Rlt strains and their metabolic properties and symbiotic features in interaction with clover. The metabolic profiling of 22 Rlt isolates with BiOLOG MicroArraysTM test was performed, comprising analyses of utilization of carbon, nitrogen, sulfur and phosphorus sources as well as tolerance to osmolites and different pH conditions. Statisti-cal analyses allowed grouping of the strains into two clus-ters: the small group comprising K4.15 and K5.4, and large of 20 remaining strains. Correlation searches between pre-viously defined genetic properties of the strains and their metabolism, revealed that strains K4.15 and K5.4 (showing similar gene distribution in the genome and lacking very large plasmids) were less sensitive to unfavorable osmotic conditions. Clovers infected by the strains lacking large plasmid (>1Mb) or possessing four plasmid irrespectively of their size had higher green wet mass of the shoots. The strain not possessing large plasmids induced more nodules on clover plants. In the studies, some relationships were observed e.g., between defined genetic properties of the strains (PCR-RFLP of rDNA ITS region, plasmid profile related to the presence of large replicons), and metabolic preferences or symbiotic affectiveness. These genetic traits can easily be observed and may be applied in the future as a screening tests for selection of strains of desired matabolic and symbiotic properties, which may be used as rhizobial inoculants. ReferencesMazur A et al. (2011) BMC Microbiology 11: 123.

P15.7

Isolation and immunochemical characterization of lipopolysaccharide from cyanobacterium Synechococcus PCC 7002Agnieszka Rombel-Bryzek1, Anna Krop-Wątorek1,2

1Department of Biotechnology and Molecular Biology, University of Opole, Opole, Poland; 2 Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Polande-mail: Agnieszka Rombel-Bryzek <[email protected]>

Lipopolysaccharide (LPS) is an integral component of the outer membrane of the cell envelope of Gram-negative bacteria, including cyanobacteria. LPS is the major viru-lence factor of Gram-negative bacteria. It is a strong stimu-lator of innate immunity in diverse of eukaryotic species. Enterobacterial LPS isolated from smooth bacterial strains consists of polysaccharide O-antigen, core oligosaccharide and lipid A which anchored entire molecule in outer mem-brane. These three parts of LPS differ in chemical struc-ture and biological properties. LPS from cyanobacteria is much less studied than many of enterobacterial LPS.The lipopolysaccharide of cyanobacterium Synechococcus PCC 7002 was obtained with hot phenol/water method and purified by ultracentrifugation. The chemical analysis of LPS preparation showed the presence of not cova-lently linked high molecular weight polysaccharide. It was removed from LPS by ultracentrifugation in the presence of detergent (Triton X-100). SDS-PAGE analysis of LPS showed the presence of fast migrating fractions only which is characteristic for rough bacteria.In order to obtain polyclonal antibodies against surface an-tigens of Synechococcus PCC 7002 intravenous immunization of rabbits with killed whole cyanobacterial cells was per-formed. The reactivity of obtained antisera was confirmed in immunoblotting test.


P15.8

Synthesis and biotransformation of chlorolactone with trimethylcyclohexene systemMałgorzata Grabarczyk

Wrocław University of Environmental and Life Sciences, Department of Chemistry, Wrocław, Polande-mail: MałgorzataGrabarczyk <[email protected]>

Hydroxylactones are often met in nature mainly in plants and sea organisms like alga or sponges. They are known for their different properties like: antimalarial (Ortet et al., 2008), antifungal (Pujar et al., 2000; Vajs et al., 1999), cyto-toxic (Zhang et al., 1997; Liu et al., 2008; El Hassany et al., 2004), antibacterial (El Hassany et al., 2004), anticancerous (El-Gamal, 2001), phytotoxicity (Fukushima et al., 1998). These compounds are usually exist in living organisms in small quantities, so their isolation is difficult and expen-sive. It is better to use methods of chemical synthesis to obtain hydroxylactones or other lactones with some func-tional groups. These last ones can be change into hydroxy-lactones using natural biocatalysts like bacteria (Liu et al., 1995, Zanaroli et al., 2006, Davis et al., 2005, Janssen, 2004) or fungi (Grotowska et al., 2002, Grabarczyk et al., 2010).A four-step synthesis and biotransrmation of chlorol-actone with trimethylcyclohexene system obtained from natural ketone alfa-ionone are presented in the paper. This chlorolactone was transformed by eight fungi strains (Fusarium species, Syncephalastrum racemosum, Botrytis cinerea) into hydroxylactone.AcknowledgementsThis project was financed by European Union from the European Re-gional Development Fund. Grant No. POIG. 01.03.01-00-158/09.

ReferencesDavis JW et al. (2005) Water Res 39: 1075-1084.El-Gamal AA (2001) Phytochemistry 57: 1197-1200.El Hassany B et al. (2004) Fitoterapia 75: 573-576.Fukushima T et al. (1998) Phytochemistry 48: 625-630.Grotowska AK et al. (2002) J Mol Catal B: Enzymatic 19-20: 203-208.Grabarczyk M et al. (2010) Biocatalysis and Biotransformations 28): 408-414.Janssen DB (2004) Curr Opin Chem Biol 8: 150-159.Liu JG et al. (2008) Phytochemistry 69: 2231-2236.Ortet R et al. (2008) Phytochemistry 69: 2961-2965.Pujar PP et al. (2000) Fitoterapia 71: 590-592.Vajs V et al. (1999) Phytochemistry 52: 383-386.Zhang J et al. (1997) Phytochemistry 46: 157-159.

P15.9

The plant-mediated enantioselective transformation of indan-1-one and indan-1-olWanda Mączka1, Aneta Szyndzielorz1, Renata Galek2, Katarzyna Wińska1

1Department of Chemistry, Wrocław University of Environmental and Life Science, Poland; 2Department of Genetics, Plant Breeding and Seed Production, Wrocław University of Environmental and Life Science, Wrocław, Polande-mail: Wanda Mączka <[email protected]>

In the last decade the need for biologically active enantiop-ure compounds for practical applications has been growing increasingly, because the way they act within living organ-isms depends largely on their absolute configuration. Chiral secondary alcohols are increasingly recognized as valuable chiral building blocks in organic syntheses of pharmaceuti-cals and agrochemicals. In the biological approaches using biocatalysts, optically active alcohols have been prepared from prochiral ketones or racemic alcohols as starting ma-terials (Itoh et al., 2008; Nakamura et al., 2001).This information encouraged us to attempt research into transformations of indan-1-one and indan-1-ol by means of enzymatic system of comminuted plant parts. The method used by us has some advantages compared to other methods, for example the very low cost, the availability of the material and the easiness of reaction work-up. To the best of our knowledge, there is no information in literature about using comminuted plant parts in transformation of those compounds.In our research we used ten biocatalysts: nine vegetables (Apium graveolens L. — celeriac, Daucus carota L. — carrot, Petroselinum crispum L. — parsley, Pastinaca sativa L. — pars-nip, Beta vulgaris L. — beet, Helianthus tuberosus L. — Je-rusalem artichoke, Solanum tuberosum L. — potato, Allium ampeloprasum L. var. porrum — leek, Raphanus sativus L. — radish) and one fruit (Malus pumila L. — apple).Indan-1-one was reduced with the excellent enantioselec-tivity (e.e. = 99.9% S(+)), but unfortunately with the low yield. The best result was obtained by means of enzymatic system of comminuted carrot, which reduced indan-1-one with yield 8.5%.All biocatalysts were able to oxidate indan-1-ol. The best result was obtained by means of enzymatic system of com-minuted Jerusalem artichoke. After 48h transformation 61% of the racemic indan-1-ol was converted to ketone, remaining the chiral alcohol in the high enantiomeric purity (e.e. = 86% R(-)).ReferencesItoh K et al. (2008) Biotechnology Letters 30: 951–954.Nakamura K et al. (2001) Tetrahedron: Asymmetry 12: 3147–3153.


P15.10

Utilization of Triton X-100 and polyethoxylated glycols during surfactant-mediated biodegradation of diesel fuelŁukasz Ławniczak1, Bogdan Wyrwas2, Alicja Szulc1, Paweł Cyplik3, Wojciech Białas3, Andrzej Szymański2, Aleksandra Hołderna-Odachowska2, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznań, Poland; 2Institute of Chemistry, Poznan University of Technology, Poznań, Poland; 3Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznań, Polande-mail: Łukasz Ławniczak <[email protected]>

The hypothesis regarding preferential biodegradation of surfactants applied for enhancement of microbial hydro-carbons degradation was studied. At first the microbial deg-radation of sole Triton X-100 by soil isolated hydrocarbon degrading bacterial consortium was confirmed under both full or limited aeration with nitrate as an electron acceptor. Triton X-100 (600 mg/L) was utilized twice as fast for aer-obic conditions (t1/2 = 10.3 hours), compared to anaerobic conditions (t1/2 = 21.8 hours). HPLC/ESI-MS analysis re-vealed the preferential biodegradation trends in both com-ponents classes of commercial Triton X-100 (alkylphenol ethoxylates) as well as polyethylene glycols. The obtained results suggest that the observed changes in the degree of ethoxylation for polyethylene glycol homologues occurred as a consequence of the ‘central fission’ mechanism dur-ing Triton X-100 biodegradation. Subsequent experiments with Triton X-100 at approx. CMC concentration (150 mg/L) and diesel oil supported our initial hypothesis that the surfactant would become the preferred carbon source even for hydrocarbon degrading bacteria. Regardless of aeration regimes Triton X-100 was utilized within 48–72 hours. Efficiency of diesel oil degradation was decreased in the presence of surfactant for aerobic conditions by ap-prox. 25% reaching 60 instead of 80% noted for experi-ments without surfactant. No surfactant influence was ob-served for anaerobic conditions. Our results are of interest to those using surfactants for biotreatment of hydrocarbon polluted sites.AcknowledgementsThis study was funded by Grant 32-194/11 DS-MK.

P15.11

Molecular analysis of Pseudomonas strains involved in oil biodegradationRobertina Ionescu, Ana-Maria Tanase, Diana Pelinescu, Csutak Ortansa, Iulia Chiciudean, Tatiana Vassu, Ileana Stoica

University of Bucharest, Faculty of Biology, Departament of Genetics, Bucharest, Romaniae-mail: ionescu robertina adriana <[email protected]>

Pseudomonas is one of the most ubiquitous bacterial group in nature and also well-known for its ability to degrade a vast set of different oil compounds - aliphatic and aromatic hydrocarbons. The aim of our study was to select Pseudomonas strains with high biodegrading abilities and capacity to produce rham-nolipids as biosurfactants and to identify them by conven-tional and molecular analyses. Our work was performed on four bacterial strains, two strains isolated from oil (named K1A, K1B) and two from oil-contaminated soil (named S7A, S8). Strains were pre-liminary identified as P. aeruginosa and P. fluorescens by con-ventional methods (Gram stain, oxidase production, pig-ment production, colony morphology, cell size and shape) and by BIOLOG system. In order to discriminate between closely related strains, ARDRA (Hae III, Msp I, Hinf I) and RAPD (BOX A1, ERIC-1R, ERIC-2) were performed. All four novel strains affiliated to P. aeruginosa and P. fluores-cens were able to grow on media having oil or n-hexadecane as sole carbon source for 21 days. Pseudomonas isolates were also screened for rhamnolipid production using drop-collapse method, determination the emulsification index (E24), oil spreading test and also tested for the presence of rhl B gene. From the four isolates, K1A strain — which proved to be affiliated at P. aeruginosa group — presented the highest bio-surfactant production and the highest capacity to degrade xenobiotic compounds and can be used for future environ-mental biotechnology.AcknowledgementsThis work has been supported by NURC project 1029/2009.


P15.12

Influence of diversity of sugar beet properties on the bioethanol productionMałgorzata Gumienna, Małgorzata Lasik, Zbigniew Czarnecki

Poznan University of Life Sciences, Institute of Food Technology of Plant Origin, Poznan, Polande-mail: Malgorzata <[email protected]>

In this study, the screening of 101 random samples of sug-ar beet has been analyzed towards the content of reducing substances, sucrose, dry matter in sugar beet pulp, as well as ethanol and reducing substances content after alcohol fer-mentation process. The influence of: nitrogen fertilization rate of sugar beets, the Optimash enzyme complex addi-tion and also the type of fermenting microorganism strain on the yield of bioethanol production has been examined.It has been found, that an average level of ethanol produc-tion is equal was 8.3 L/100 kg of sugar beet pulp.Nitrogen fertilization has had a significant effect on etha-nol efficiency, best result has been achieved with 120 kg/ha, whereas doses of 160 kg/ha and 80 kg/ha have lowered the efficiency of fermentation process.The highest fermentation rate has been achieved with use of yeast strains of Saccharomyces compared with bacteria strain Z. mobilis. Both Fermiol and Safdistil yeast strains have had comparable fermentation efficiency. The Opti-mash enzyme complex addition has had a positive effect on fermentation rate, where dose equal to 50% of the dos-age suggested by the producer, has resulted in significant lower ethanol yield.

P15.13

The evaluation of selected corn strains as raw material for bioethanol productionMałgorzata Lasik, Małgorzata Gumienna, Jack Nowak, Zbigniew Czarnecki

Poznan University of Life Sciences, Institute of Food Technology of Plant Origin, Poznan, Polande-mail: Zbigniew <[email protected]>

The aim of the presented work constitute the evaluation of selected corn strains potential for bioethanol produc-tion and the impact of technological modifications on the efficiency of the process.260 of corns samples, races and hybrids were investigated in the experiment. Each sample has been examined for the raw material chemical characteristic and the ethanol fer-mentation efficiency using non pressure starch liquefaction method.The sample witch demonstrated the highest bioethanol yield has been assigned for further analysis composed of influence of the fermentation method, the yeast prepa-ration, the density of milled raw material and the addition of Optimash VR enzyme on the efficiency of the ethanol production.Three different dried yeast Saccharomyces cervisiae prepara-tions were examined: Fermiol, Safdistil and As4. The exper-iments were performed with the use of two fermentation methods: non pressure starch liquefaction and simultane-ous saccharification and fermentation (SSF). It was found, that Fermiol indicated the highest fermentation efficiency. Comparing the two examined methods, the non pressure technique showed significantly higher ethanol yield. Simi-larly, evaluating the influence of Optimash VR enzyme addition, best results were observed when non pressure starch liquefaction method was performed.


P15.14

Proteomic response of roots in Salix viminalis L. grown on various tannery wasteAgata Zemleduch, Gabriela Lorenc-Plucińska

Institute of Dendrology, Polish Academy of Sciences, Polande-mail: Agata Zemleduch <[email protected]>

The global tanning industry generates up to 4 million tonnes of solid waste per year. Polish tanneries produce an-nually about 250 t. The waste varies in physical and chemi-cal properties depending on tanning technology or sludge treatment. However, generally the final waste is an or-ganic solid matrix, composed of by-products from leather processing, and containing various amounts of heavy met-als, particularly chromium. Landfilling is the main way of waste disposal but it may have serious consequences since it can cause pollution of groundwater, surface water, and soil. The common technologies of land remediation turn out to be complex and expensive. Thus bio-technologies with the use of green plants are proposed as an effective al-ternative. Phytoremediation is a non-invasive and low-cost technique. It is based on the natural capability of plants to take up potentially harmful chemicals, e.g. heavy met-als, into their tissues or to stabilize them in the ground. Moreover, tannery waste is rich in organic and inorganic components that can potentially stimulate plant growth. Their agronomic use as amending agents is still hindered by the scarcity of studies on the ecological effects of tannery wastes and the abundance of reports on the toxic effect of Cr-rich wastes on plant development, growth and physiol-ogy. The use of trees from the family Salicaceae (not edible but energy crops) in phytoremediation makes it possible to combine safe and effective remediation strategies with biomass production. This is because they are characterized by fast growth, high productivity, and are confirmed to be generally tolerant to many abiotic stresses in the environ-ment.We studied the ability of willows and poplars to grow on solid tannery waste varying in origin, different time of dis-posal, as well as physical and chemical parameters (pH, conductivity, organic matter) and mineral content (P, N, Ca, Mg, K, Fe, Cr). Here we present some morphometric re-sults showing a distinct response of Salix viminalis L. grown on fresh and old tannery waste. Although roots of plants in both experimental variants have a similar percentage of changed proteins in proteomic profiles (about 10%, com-pared to control roots), it seems that different proteins may be involved in metabolic adaptation during growth on both kinds of wastes. The use of Salix viminalis, which is a close relative to the model tree Populus trichocarpa, could enable us to find some protein markers that can help in selection of best specimens for phytoremediation.

P15.15

The role of the Thr-65 in the catalysis of catechol 2,3-dioxygenase from Planococcus sp. S5Katarzyna Hupert-Kocurek, Agnieszka Stawicka, Danuta Wojcieszyńska

University of Silesia, Faculty of Biology and Environmental Protection, Department of Biochemistry, Katowice, Polande-mail: Katarzyna Hupert-Kocurek <[email protected]>

Ring cleavage dioxygenases are the key enzymes in aromat-ic structure biodegradation. These enzymes,catalyzing the addition of two atoms from molecular oxygen into the aro-matic ring of catechols and substitued catechols, are classi-fied into two groups: intradiol dioxygenases and extradiol dioxygenases. Intradiol enzymes incorporate two atoms of the oxygen molecule between the vicinal hydroxyl groups while the extradiol dioxygenases cleave the aromatic ring of substrate outside the two hydroxyl groups and produce muconic semialdehyde. Activity of extradiol dioxygenases depends on the presence of a Fe(II) ion, that is bound to two histidine and one glutamate residues. Catechol 2,3-dioxygenases (C23Os) are enzymes belong-ing to the extradiol dioxygenases family commonly found in different bacteria species including Acinetobacter, Bacillus, Pseudomonas, Rhizobium and Sphingomonas.Gram positive strain Planococcus sp. S5 is able to grow on various aromatic compounds such phenol, salicylate and benzoate, as the sole source as carbon and energy. c23o gen coding for catechol 2,3-dioxygenase from this strain was mutagenized and cloned into Escherichia coli. In this studies, expressed in Escherichia coli, wild type and mutated catechol 2,3-dioxygenase of Planococcus sp. S5 was isolated and characterized. The catalytic parameters of C23Owt and the mutant C23OB67 were studied on cat-echol as a function of pH and temperature and by using three catechol derivatives: 3-methylcatechol, 4-methylcate-chol and 4-chlorocatechol as substrates. The results obtained show that substitution of single ami-no acid (Met65Thr) affected enzyme activity and substrate specificity as well as kinetic parameters of catechol 2,3-di-oxygenase. Characterization of mutant protein showed that the maximum activity was obtained at 35°C and pH 4.0 while pH optima of the wild type enzyme expressed in E. coli was 7.0. The mutant C23OB67 had higher affinity for catechol. The value of Km for this substrate with mutant protein (9.05 µM) was lower than that determined for wild type catechol 2,3-dioxygenase. Mutant protein showed rela-tive activity for the following catechols: catechol (100%), 3-methylcatechol (7%), 4-methylcatechol (124%), 4-chloro-catechol (217%). The activity of C23OB67 towards 4-chlo-rocatechol was significantly higher from that observed for C23Owt.


P15.16

Indices of oxidative stress in lead (Pb) exposed Talinum cuneifolium (Ceylon spinach, Portulacaceae) — Relevance to phytoremediationAbhay Kumar, M. N. V. Prasad

University of Hyderabad, Department of Plant Sciences, Hyderabad, India.e-mail: Abhay Kumar <[email protected]>

Lead (Pb) introduces in to the environment through min-ing, smelting of ores, automobile exhaust, sewage sludge, coal burning, Pb containing paints, manufacture of bat-teries, bullets and microelectronics. Pb has no metabolic significance and is hazardous to humans, plants and other biota. A numbers of plants are reported to accumulate the Pb from contaminated media and are useful for risk assess-ment studies. Talinum cuneifolium is a terrestrial leafy vegeta-ble. Species of Talinum are useful for heavy metals moni-toring. The aim of the study is to examine the ability of T. cuneifolium for Pb accumulation and to examine the effect of Pb on it. Hydroponic experiments were conducted with stem cuttings using modified Hoagland’s solution. These stem cuttings in hydroponics were treated with 0, 0.25, 0.50, 0.75, 1.0 and 1.25 mM of Pb(NO3)2 for 7 days. Pb exposed twigs were analyzed for Pb accumulation, protein content, protein oxidation, activity of antioxidative enzymes [su-peroxide dismutase (SOD), catalase (CAT)], glutathione, glutathione reductase (GR) enzymes and thiols content in roots. Our results showed that Pb accumulation in adventi-tious roots increased as a function of the external Pb con-centration. Reduction in protein content was observed and is directly related to metal concentrations. The carbonyl contents were found to increase in all treated roots in com-parison to control, while a reduction in protein has been observed. Seven days treatment with different dose of Pb caused an increase in the activities of antioxidative enzymes such as SOD and CAT in dose dependent manners, which explain antioxidative property of the plant. Glutathione is one of the major cellular safeguards. Ratio of reduced and oxidized glutathione (GSH/GSSG) decline when Pb con-centrations increased, this is evidently due to reduction in GSH biosynthesis or rapid reduction of GSSG by the en-zyme GR. Similarly, GR enzyme activity also increased only at 0.25 mM of Pb and decreased (not significant) at 0.75- 1.25 mM of Pb. Decline in GSH at higher dose of Pb treat-ment be attributed to rapid consumption rate of GSH as antioxidant that is required for the synthesis of phytoche-latins, a thiol containing bioligand, a well established heavy metals detoxification mechanism. In terms of non-protein thiols the concentration is significantly (P ≤ 0.05) decreased at all Pb treatment except 0.25 mM of Pb, which supports the synthesis of metal chelating thiols at 0.50–1.25 mM Pb. These findings are discussed for their relevance to bioindi-cation, biomonitoring and for phytoremediation.

P15.17

Application of microscopic fungi for quinoline and its derivatives eliminationAleksandra Felczak, Katarzyna Lisowska

Department of Industrial Microbiology and Biotechnology, University of Lodz, Lodz, Polande-mail: Felczak Aleksandra <[email protected]>

Quinoline and its derivatives are N-heterocyclic aromatic compounds, which naturally occur in coal tar and oil. Qui-noline serves as an intermediate in the production of vari-ous compounds, including fluoroquinolone antibiotics (e.g. ciprofloxacine). Published data clearly indicate that quino-line and some of its derivatives have toxic, mutagenic and carcinogenic properties. Thus, widespread contamination of environment by quinolons has become a great concern. In the recent years, degradation of quinoline by bacterial strains has been described, but there is still no informa-tion about the ability of fungi to remove the mentioned compounds.The aim of the presented work was to determine the pos-sibilities of using microscopic fungi for quinoline and cip-rofloxacine elimination. The studies were performed with 5 strains of filamentous fungi, which are known for their ability to degrade persistent organic pollutants. Among the tested microorganisms, only 2 fungal strains (Curvularia sp and Cunninghamella elegans) exhibited good tolerance to quinoline. The mentioned strains also displayed an ability to remove quinoline. The efficiency of quinoline utiliza-tion, in samples containing 250 mg/l quinoline, achieved the value of 60% and 50% for Curvularia sp and C. elegans cultures, respectively. The studies conducted with the use of ciprofloxacin indicated that both strains were capable of growing in the presence of antibiotics. Additional analy-ses revealed a considerable depletion of ciprofloxacin in the fungal cultures. The presented results allowed selecting strains capable of growth and efficient elimination of qui-noline, which opens new possibilities for further research on biotransformation of fluoroquinolones.


P15.18

Influence of alkyl polygycosides on diesel oil biodegradationKarina Sałek, Ewa Kaczorek, Nina Pisarek, Andrzej Olszanowski

Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Polande-mail: Karina Sałek <[email protected]>

Biodegradation of organic compounds including diesel oil and its derivatives plays an important role in the environ-mental protection. However, that process can be limited by a small or even lack of solubility of hydrocarbons in water. This is why the use of surfactants in order to enhance the remediation processes becomes more and more popular. Among those surface active agents alkyl polyglycosides (APG) gain a growing popularity. It has been proved that these surfactants can easily undergo biodegradation, but can also help bacterial degradation of well known pollut-ants like fuels etc. Moreover, APGs can be obtained from natural, renewable sources which make them perfect for food and cosmetic industry as well as for bioremediation processes as described above. Alkyl polyglycosides belong to the class of nonionic surfactants. They consist of sugar (glucose) and fatty alcohol. The aim of this work was to assess the influence of se-lected alkyl polyglycosides on biodegradation of diesel oil as well as their influence on hydrophobicity of chosen bac-terial strains. Surfactants used in our works were: Glukapon 215, Glukapon 650 and Lutensol GD 70. Environmental gram-negative strains used in our analyses were: Stenotropho-monas maltophilia and Achromobacter denitrificans. Both tested strains have strictly hydrophilic features and addition of surfactants does not influence them much. In order to observe potential changes in hydrophobicity of microbial cells in different systems with diesel oil, two oth-er systems with bacteria were tested. These were: “bacterial strain-diesel oil” and “bacterial strain-diesel oil-surfactant”. Obtained results indicated that the hydrophobicity of both Stenotrophomonas maltophilia and Achromobacter denitrificans strains was higher in system “strain-diesel oil” when com-pared to two systems with surfactants. When it comes to biodegradation, both of chosen strains indicated higher biodegradation degree in systems “strain-diesel oil-surfactant” in comparison to those without addi-tion of surfactants. What is more, biodegradation of diesel oil did not differ much among tested surfactants as well as their concentrations. This means that the growth of sur-factants’ concentrations did not influence the degree of biodegradation in a significant way. AcknowledgementsThis study was supported by Grant No. N N304 163337 Polish Ministry of Science and Higher Education.

P15.19

Application of advanced oxidation processes (AOP`s) for the industrial wastewater treatmentDorota Krzemińska, Ewa Neczaj, Anna Grosser

Częstochowa University of Technology, Institute of Environmental Engineering, Częstochowa, Polande-mail: Krzemińska Dorota <[email protected]>

Integration of advanced oxidation processes (AOPs) and other traditional wastewater treatment technologies has been proven to be more effective for treating industrial wastewater economically. Industry produces large quanti-ties of highly polluted wastewater containing toxic sub-stances, organic and inorganic compounds such as:heavy metals, pesticides, phenols and derivatives thereof, aromat-ic and aliphatic hydrocarbons, halogenated compounds, etc., which are generally resistant todestruction by biologi-cal treatment methods. Different physical, chemical, and biological treatment processes have been employed to treat various industrial wastewaters such as food, pharmaceuti-cal, pulp and paper, dye processing and textile, and land-fill leachate effluents. The research showed that the AOP process can be considered as an interesting option for the depollution of toxic and poorly biodegradable industrial wastewater. Advanced Oxidation process include: the Fen-ton reaction, UV photolysis, sonication, ozonation, elec-trochemical oxidation, etc.. These processes involve the generation of highly free radicals, mainly hydroxyl radical (HO•) via chemical, photochemical and photocatalytic re-actions. All these methods have a high potential for redox so that they are capable of oxidizing many organic com-pounds. The way of selecting the AOP methods depends on the characteristics of the waste stream, environmental regulations, and cost. Reviewing the experimental works on this area and discussing their effectiveness would be helpful for deciding whether the integrated processes is effective to fulfill the annually restricted legislations with lower invest-ment. The article presents the possibilities of the use of advanced oxidation methods and their combination with biological treatments for industrial wastewater decontami-nation. It was shown that integration of advanced oxida-tion process and biological treatment methods are useful in order to fulfill the environmental regulations.


P15.20

The determination of PME and RME biodiesels’ properties produced from the used palm and rape-seed oils as well as of methanol biodegradation in output wastewatersPrzemysław Petryszak1, Barbara Kulig1, Grzegorz Wcisło2, Henryk Kołoczek1

1University of Agriculture in Kraków, Faculty of Horticulture, Department of Biochemistry, Kraków, Poland; 2 University of Agriculture in Kraków, Faculty of Production Engineering and Power Engineering, Department of Power Engineering and Automatization of Agricultural Processes, Kraków, Polande-mail: Przemysław Petryszak <[email protected]>

The issue related to the possibility of making use of waste raw materials for biofuels production attracts a lot of atten-tion nowadays. Plant oils and animal fats are basic materials as regards the production of FAME Biodiesel (Fatty Acid Methyl Esters).Waste oils coming from food industry have aroused a lot of interest recently. As for Europe, rape-seed oil and palm oil are most often used products for culinary purposes. The poster presents the results of research de-termining physicochemical properties of PME (Palm Oil Methyl Esters) and RME (Rapsod Methyl Esters) Biofuels obtained from palm oil used for chip (French fries) frying and from rape-seed oil used for fish frying. PME and RME biofuels were produced in a GW-10 reactor constructed by one of the authors. Methyl alcohol was used for transes-terification of the oils with alkaline potassium hydroxide KOH used as a catalyst. The process of transesterification was carried out in two stages and the obtained degree of oils conversion into esters was equal to over 98% (m/m). The examined biofuels satisfy the requirements of EN 14214 standard of a Diesel engine as regards the content of esters in FAME. The determination of the conversion degree was conducted in compliance with EN ISO 5508 using a THERMO Scientific GC Ultra type. On the basis of the obtained results it can be stated that both FAMEs contain a big amount of useful oleinic acid esters. It should be stressed that in PME biodiesel there are more esters, more than 20 % compared to the esters contained in RME. For both produced biofuels physicochemical parameters, such as the viscosity coefficient, the cetane number, the heat of combustion and the calorific value were measured. The implementation of PME and RME production on a large scale depends, except technical parameters, on the way the problem of waste waters containing toxic metha-nol is solved. The obtained results have proved that Me-tOH contained in waste waters from RME biofuel produc-tion is fully degraded by developed microbial consortium. In the case of MetOH in PME waste waters the process of degradation is slower and within 20 days of biodegrada-tion the MetOH concentration is reduced by about 30%. The obtained results eliminate the waste waters from being processed in biological sewage treatment plants. Researches into the selection of special microorganisms being able to effectively degrade MetOH in PME waste waters are being carried out at present.

P15.21

Biodegradation of alkyl derivatives of aromatic hydrocarbons by Pseudomonas stutzeri strainsEwa Kaczorek, Karina Sałek, Bartosz Lis, Andrzej Olszanowski

Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznan, Polande-mail: Ewa <[email protected]>

Environmental pollution by aromatic hydrocarbons is a serious ecological problem. There are many various clean-up technologies, which can be categorized in three general schemes: chemical, physical and biological. The use of bio-technology, especially bioremediation allows for high de-grees of purification of contaminated sites. Hydrocarbons, due to their low water solubility have limited possibilities, enhanced by microorganisms. Surfactants enhance the re-mediation of hydrophobic compounds by increasing hy-drocarbon solubilization or mobilization of hydrocarbon from soil into aqueous phases.There are many types of aerobic and anaerobic micro-organisms capable of degrading aromatic hydrocarbons. Among them there is a dominant group of bacteria of the Pseudomonas genus. These are gram-negative microorgan-isms leading metabolism under aerobic conditions, and are characterized by a wide tolerance to different environmen-tal factors. An example of the type strain of Pseudomonas biodegradation activity is Pseudomonas stutzeri. Literature reports indicate that the degradation of aromatic hydrocar-bons is initiated by the connection of the oxygen particles to the aromatic ring. This process is possible due oxidore-ductase enzymes produced by the bacterium.The aim of this study was to determine the surface proper-ties of two strains of Pseudomonas stutzeri and analysis of their impact on the biodegradation of of alkyl derivatives of aromatic hydrocarbons. Moreover, the influence of bio-surfactant rhamnolipides on biodegradation process was tested.The study has shown significant changes in the cell surface for tested bacterial strains. Modification of bacterial cell surface depends on the type of carbon source used in the experiments or the presence of surfactant. The conducted processes of biodegradation have proven the efficiency of all strains of Pseudomonas stutzeri in the reduction of pollu-tion by microorganisms, achieving the degradation of com-pounds level of over 50%.The study has also revealed the differences between the examined strains of Pseudomonas stutzeri, which, despite be-ing the same species, have different cell surface properties.AcknowledgementsThis study was supported by Grant No. N N304 163337 Polish Ministry of Science and Higher Education


P15.22

EMA-real-time PCR as a method in the diagnosis of pathogens in sewage treatment processMałgorzata Kacprzak, Krzysztof Fijałkowski, Agnieszka Rorat

Institute of Environmental Engineering, Częstochowa University of Technology, Częstochowa, Polande-mail: Agnieszka Rorat <[email protected]>

One of the main reasons that limits the usage of sewage sludge in agriculture are microorganisms which may cause many diseases e.g. Escherichia coli 0157:H7. Traditional di-agnosis methods are very costly and lengthy so new tech-niques are needed. Real-time PCR is a quick, easy molecular biology tool and it allows to mark all known bacteria in few hours. However it doesn’t give a chance to distinguish live from dead bacteria. It has been proved that ethidium bro-mide monoazide (EMA) is a dye which may penetrate only to dead cells and bind to their DNA. That is why it may be used in the quick diagnosis tests in samples like sewage sludge or sludge.DNA was isolated from different stages of wastewater treatment with the usage of EMA and without it. Then real-time PCR was used to indicate the amount of cells of Escherichia coli and Salmonella typhimurium in samples, with and without EMA pretreatment. Petri dish method was also used to compare with the results from real-time PCR method. The obtained results shown that ethidium bro-mide monoazide may be used in DNA isolation to prevent false positive results.

P15.23

Adsorption and bioregeneration in the treatment of phenol with activated carbonMagdalena Madeła, January Bień, Ewa Neczaj

Institute of Environmental Engineering, Technical university of Czestochowa, Czestochowa, Polande-mail: Magdalena Madeła <[email protected]>

Phenols as well as their secondary compounds can be ef-fectively removed on activated carbon. In literature process of biological regeneration is widely described and has fun-damental significance in the case of activated carbons used in water conditioning processes and wastewater treatment.This paper presents results of the investigation on sorption and biodegradation of phenol with activated carbons. In this study, a waste water model was used at phenol concen-trations of 600 mg/dm3. Two types of activated carbons: ROW 08 Supra and WG-12 were used in the experiment. The main difference between those carbons is bulk density. The carbon WG-12 has a reported density of 26.1 lb/cu ft. The carbon ROW 08 Supra is lighter and has reported density of 23.63 lb/cu ft. Taking into consideration capil-lary disintegration, it was possible to affirm, that WG-12 is typical agent of microporous carbons, however carbon ROW 08 SUPRA — mezoporous carbons.The adsorption process was carried out by the static meth-od. The aim of this work was to determine the Freundlich isotherm. It was prepared with the following procedure: 250 cm3 of phenol solutions, were mixed with 1 g of acti-vated carbon previously dried in temperature 145°C. The phenol concentration in water after adsorption was deter-mined by the spectrophotometric method.The process of bioregeneration was carried out under aerobic conditions. The bioproduct marked as DBC Plus R-5 was used for phenol biodegradation from surface of activated carbon. It is biological compound used in treat-ment of industrial wastewater, selective for biodegradation of phenols. Bioproduct concentration 1 g/dm3 was used. Varied contact time of activated carbons with bioproduct — 4, 8 and 24 h was estimated.Regenerated samples of carbon were again adsorbed by phenol from solution, also at concentration 600 mg/dm3. Adsorption capacity of regenerated active carbon was de-termined. Degree of regeneration was counted as relation of adsorption capacity of regenerated carbon in relation to adsorption capacity of fresh carbon.On the basis of obtained results it was affirmed that car-bon WG-12 shows better adsorption abilities for phenol removing from water in comparison with carbon ROW 08 Supra. The results also show, that degree of regeneration depends on contact time of activated carbon with bioprod-uct. Result of sorption before and post phenol biodegrada-tion showed that regeneration factor was equal about 75%.


P15.24

Violaxanthin and diadinoxanthin de-epoxidation in model lipid systemsDariusz Latowski1, Reimund Goss2, Kazimierz Strzałka1

1Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 2Institute of Biology I, University of Leipzig, Leipzig, Germanye-mail: Dariusz Latowski <[email protected]>

De-epoxidation is light dependent step in all known kinds of xanthopyll cycles which are important photoprotective processes functioning in plants. In vascular plants violax-anthin (Vx) cycle occurs which involves interconversion between: Vx, antheraxanthin (Ax) and zeaxanthin (Zx). These reactions are catalyzed by violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase. In diatoms operates an-other kind of the xanthophyll cycle, diadinoxanthin (Ddx) cycle in which interconversion between Ddx and diatoxan-thin (Dtx) takes place. Enzymes catalyzing these reactions are diadinoxanthin de-epoxidase (DDE) and diatoxanthin epoxidase. In the present studies the influence of lipids on the de-epoxidation of the xanthophyll cycle pigments, Vx to Zx as well as Ddx to Dtx was investigated. Experiments on the significance of physical properties of the aggregates formed by inverted lipid micelles, which are necessary for de-epoxidation, on the conversion of Vx to Zx via Ax and Ddx to Dtx were performed. Dependence between the conversion of Vx into Ax and Zx as well as Ddx to Dtx and the molecular dynamics of hydrophobic fraction of aggregates formed by inverted micelles were studied. Thickness of the hydrophobic fraction of the aggregates was another tested parameter. In this experiment two types of PE, esterified with fatty acids having one double bond but differing in length were used. Size of the inverted micelles, suggested by mathematical description of the structures, was the last tested parameter. Diameters of the micelles varied from 7 to 9 nm, when they were created by MGDG, about 15 nm and from 20 to 21 nm when mixtures of MGDG and DGDG (50:50 and 85:15, respectively) were used. The obtained results show that the rate of de-epoxidation strongly depends on all tested physicochemical properties of lipids. Mutual orientation of enzyme and substrate mol-ecules and dilution of pigments by lipids are postulated as main mechanisms to explain the results.

P15.25

Zeaxanthin epoxidation in semi-defined model systemPaulina Kuczyńska1,2, Dariusz Latowski1, Sylvia Niczyporuk3, Monika Olchawa-Pajor1, Peter Jahns3, Wiesław I. Gruszecki4, Kazimierz Strzałka1

1Department of Plant Physiology and Biochemistry, Jagiellonian University, Kraków, Poland; 2Department of Plant Physiology, Institute of Biology, Pedagogical University, Kraków, Poland; 3Plant Biochemistry, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany; 4Marii Curie-Skłodowskiej 1/201, 20-031 Lublin, Polandle-mail: Paulina Kuczyńska <[email protected]>

The most common type of xanthophyll cycle is violaxan-thin cycle which occurs in higher plants, mosses, lichens and some algae and protects them against overexcitation. In the violaxanthin cycle are two reversible reactions. De-epoxidation of violaxanthin (Vx) which has two epoxy groups is catalysed by violaxanthin de-epoxidase (VDE). In the reverse reaction zeaxanthin (Zx), an epoxy-free pig-ment is conveted to violaxanthin and it is catalysed by ze-axanthin epoxidase (ZE), an enzyme located on the stromal side of thylakoids. NADPH, FAD, molecular oxygen and pH 7.5 are required to zeaxanthin epoxidation. There exist model systems to study Vx de-epoxidation with the use of isolated enzyme. Such approach in the case of Zx epoxida-tion is difficult because the respective enzyme has not been isolated and purified by now. This study deals with devel-opment of a model system of Zx epoxidation. Two assay systems are presented in which reduction in the Zx amount was observed. In these assays two mutants of Arabidopsis thaliana which have active only one of the two xanthophyll cycle enzymes were used. Npq1 mutant produces ZE and it may convert zeaxanthin to violaxanthin. However, the VDE is inactive and plants are sensitive to strong light con-ditions. The other mutant, npq2, produces VDE and con-verts Vx to Zx under strong light but reverse reaction is not possible. This mutant is good source of Zx because it can accumulate this pigment. The first assay containing thylakoids from npq1 and npq2 mutants of Arabidopsis thal-iana gave positive results and high efficiency of epoxidation reaction was observed. The amount of Zx was reduced by 65%. To optimize high efficiency of this reaction there were studied factors facilitating both fusion of the two types of thylakoids and the optimal time of sonication is 7 seconds with 3 repeats. The second parameter studied in this assay system was the ratio of thylakoids from npq1 and npq2 and the best was 1:1 where were observed the biggest decrease of Zx level. The amount of thylakoids determined by chlorophylls concentration in reaction mixture was also important. The second kind of assay contained thylakoids npq1 mutant of A. thaliana because it has a capability of Zx epoxidation and exogenous Zx with MGDG. Study chang-es in the ratio of Zx and MGDG showed that the optimal was 1:60. In this system, due to epoxidation, the amount of Zx was reduced by 54% of its initial level. This in vitro system of Zx epoxidation enables analysis some properties of the enzyme without necessity of its isolation.


P15.26

Stabilization of heavy metals in acidic soil with sewage sludge, inorganic amendments and fescue (Festuca arundinacea Schreb. ) growthMałgorzata Kacprzak, Anna Grobelak

Czestochowa University of Technology, Institute of Environmental Engineering, Czestochowa, Polande-mail: Malgorzata Kacprzak <[email protected]>

Heavy metals are common environmental contaminants in industrialized regions. The long term heavy metal deposi-tion causes their accumulation on a surface area and migra-tion into soil profile. The trace elements may be leached into groundwater, exported with erosion into surface water or may enter food chains. The areas covered by the activi-ties of steel industry, metallurgical industry and coal fired plants are also contaminated with anion excess for exam-ple sulfur anions, what increases the metal toxicity due to a soil strong acidification. Traditional remediation options are coming under increasing criticism for their high costs, energy intensiveness, site destructiveness and public dissat-isfaction. Contaminated sites generally support little veg-etation, therefore the induced phytostabilization methods seems to be justified. The main objectives of the present study were to determine the effect of sewage sludge and different inorganic amendments (lime, triple superphos-phate, potassium phosphate) on the process of improved Cd, Pb and Zn phytostabilization in the cultivation of Festu-ca arundinacea Schreb. and to examine heavy metal bioavail-ability in amended soil. A pot assessment was conducted in the growth chamber using contaminated soil from the site surrounding thezinc smelter in Miasteczko Slaskie Silesia region in Poland. Soil in the study area was contaminated with heavy metals, especially Cd (14 mg/kg), Pb (1400 mg/kg) and Zn (1000 mg/kg). Raising pH and supplementing with fertilizers (influencing the phytoavailability of metals) made this soil suitable for fescue growth. The concentra-tions of bioavailable heavy metals decreased significantly especially for combination of 0.8% CaO, 0.8% potassium phosphate and 1% d.m. of sewage sludge. In control soil after 8 weeks of experiment the concentrations ofbioavail-able heavy metals were very high (Cd 7.6 mg/kg, Pb 74 mg/kg, Zn 117.4 mg/kg) compared to the described treat-ment (Cd 0.075 mg/kg Pb 0.16 mg/kg, Zn 2.34 mg/kg).Application of sewage sludge and inorganic compounds caused decrease of the total concentration of all investigat-ed heavy metals in plant tissues. All amendments influenced the plant biomass, however the highest yield was achieved after combined treatment. The risk of metal toxicity to the food chain can be reduced by phytostabilization process. The combination of sewage sludge, lime, phosphorus fer-tilizers and fecue grasses seems to be the most effective.

P15.27

Enzymatic lactonization of cis- and trans-epoxyesters with cyclohexane ringMałgorzata Grabarczyk, Teresa Olejniczak

Wrocław University of Environmental and Life Sciences, Department of Chemistry, Wrocław, Poland e-mail: Małgorzata_Grabarczyk <[email protected]>

Lactones are major contributors to the characteristic fla-vour of many natural food products (Cooke et al., 2009). Some of terpenoids lactones have antifungal (Carda et al 2003) or cytostatic activity (Euzebio et al., 2009). The analy-sis active lactones proofs that slight differences in structure are changing the smell or taste, those effects are also con-cerning the change of the configuration of the stereogenic centres.We are interested in receiving pure enantiomers of lactones by chemoenzymatic syntheses. In our earlier experiments we use whole-cell microorganisms as biocatalysts of lac-tonization of epoxy esters (Olejniczak et al., 2001).Actually we try applied commercially available enzymes for biotransformation cis- and trans-ethyl 2-(4,4-dimethyl-2,3-epoxycyclohexyl)acetate or cis- trans-ethyl 2-(6,6-dimethyl-2,3-epoxycyclohexyl)acetate in obtaining optical active lac-tones.Before the biotransformation, the structures of epox-yesters were established. Two different mechanisms of lactonization of cis- trans-epoxy esters in water conditions were proposed. The mechanism of microbial catalysis (with the expected activity of of epoxide hydrolases, (en-zymes which enantioselectively open epoxide ring to diols) was compared with the enzymatic preparations (lipases, esterases) which catalyze ester bond hydrolysis or intramo-lecular transestrification.It was observed that the course of transformation is dif-ferent for cis and trans-epoxyesters. Five out of 37 tested biocatalysts were capable of biotransformation leading to (+)-or (-)-enantiomers of epoxyesters and hydroxylactones. Pure enantiomer of were isolated after transformated by lipase from Candida antarctica, Rhizomucor miehei, Mucor java-nicus.ReferencesCooke RC et al. (2009) J Agric Food Chem 57: 2462-2467.Carda M et al. (2003) Tetrahedron 59: 857-864.Euzebio FPG et al. (2009) Bioorganic Chem 37: 96-100.Olejniczak et al. (2001) Chirality 13: 302-307.


P15.28

Phenolic plant compounds stimulate biotransformation of monochlorophenolsIzabela Greń1, Magdalena Kurek2, Danuta Wojcieszyńska1, Joanna Bugielska1

1University of Silesia, Department of Biochemistry, Poland; 2Silesian University of Technology, Department of Environmental Biotechnology, Polande-mail: Greń <[email protected]>

Chlorophenols belong to the group of xenobiotics which removing from the natural environments is difficult. Structure of the aromatic ring with the hydroxyl group and neighboring chloride substituent is very resistant to chemical as well as microbial degradation. Chlorophenols rarely support the growth of microorganisms and they usu-ally exhibit the exceptional toxicity to the living organisms. Cometabolic degradation is nowadays thought to over-come this problem because although cometabolite doesn’t serve as the source of carbon and energy for microorgan-isms, the presence of additional source of carbon help to overcome this problem. Biodegradation of easily degraded source of carbon gives energy and the reduction factors which are used for the cometabolite transformation.Gram-negative Stenotrophomonas malthophilia KB2 is able to grow on various aromatic compounds such as phenol, cresols, benzoate or some its derivatives as the sole source as carbon and energy. Some of these compounds are of plant origin, e.g. 4-hydroxybenzoate (4-HB), 3,4-dihydroxy-benzoate (DHB) or vanillate (VAN). These compounds which enter the environment by exudation or are secreting directly into the soil after decay of plants might be used as natural substrates to improve xenobiotic degradation rate.Biodegradation of 1 mM monochlorophenols (MCP) iso-mers in the presence of 3 mM 4-HB, DHB or VAN as the growth substrates (GS) by phenol-pregrown cell of KB2 strain was studied. Irrespective of kind of the growth-substrate used in the cometabolic cultures, Stenotrophomonas malthophilia KB2 metabolized more effectively monochlo-rophenols in the comparison with the cultures where MCPs were introduced as the only source of carbon and energy. Complete degradation of 4-chlorophenol was observed in the presence of protocatechuic acid, which also stimulated biotrasformation of 2-CP and 3-CP the most effectively. The highest biotransformation rate of 3-chlorophenol was observed while vanillic acid was introduced as the growth substrate. Usage of phenolic compounds naturally found in soil and water for removing the xenobiotics from the environment seems to be good alternative for common-ly exploited so far phenol as the growth substrate in the cometabolic cultures.

P15.29

Comparison of ARDRA, REP-PCR and microscopic analysis as methods in early bulking activated sludge symptoms detectionDagna Sołtysik, Daria Blaszczyk, Ilona Bednarek

Department of Biotechnology and Genetic Engineering, Medical University of Silesia, Polande-mail: Dagna Sołtysik <[email protected]>

Activated sludge process is one of the most popular and widest used microbiological technology in wastewater treatment plants. Efficiency of treatment process depends mostly on sludge flocks settleability. Overgrowth of fila-mentous bacteria affect flocks settlement causing incorrect sedimentation and separation of microbiological biomass from effluent stream. This observed unwanted effect is called bulking of activated sludge. Temporary changes in bacterial community structure are monitoring in WWTPs by microscopic observation. However, literature data sug-gest that molecular methods seems to be more accurancy and effective. The aim of this study was to compare the utility of microscopic observation and population finger-printing patterns obtain by amplified ribosomal DNA re-striction analysis (ARDRA) and ribosomal extragenic pal-indromic PCR, in early bulking symptoms detecting.Active sludge samples, from WWTP in Łaziska Górne, were collected during 19 months, once or twice per month. Immediately after collection, physicochemical and mi-croscopic analyses (according to the Polish Standards for wastewater) were performed. Isolated genomic DNA was used as a template molecular analysis. To obtain sludges DNA fingerprints by ARDRA technique, amplification of 16S rRNA genes fragment with universal primers 27F and 1492R, and following restriction analyses of 16S rRNA amplimers by means of choosen endonucleases, were car-ried out. To receive fingerprinting patterns by REP-PCR analysis primers complementary to conservative regions in REP-elements were used. Basing on numbers and position of bands on electrophoresis gel, obtained from ARDRA and REP-PCR analyses of each sludge samples, dendro-grams using the Ward’s method were created.Environmental conditions and substrate concentration play an important role in bulking sludge development, which unfavorable process may be identify earlier by means of molecular techniques. Dendrograms allow to classify samples of activated sludge to non-bulking or bulking. Variations of fingerprinting patterns obtained using both molecular methods point at permanent transformation of activated sludge microbial community. Comparison of mo-lecular and microscopic method result in confirmation that ARDRA and REP-PCR may be useful methods for detec-tion of the earliest microbial community changes, leading to bulking episodes.AcknowledgementsThis study was financially supported by research grant KNW-1-036/P/1/0, subsidized by Medical University of Silesia.


P15.30

Utilization of rhamnolipids and its effects on population of diesel and biodiesel degrading bacteria under various aeration conditionsŁukasz Chrzanowski1, Mariusz Dziadas2, Marcin Schmidt3, Łukasz Ławniczak1, Paweł Cyplik3, Wojciech Białas3, Alicja Szulc1, Piotr Lisiecki1, Henryk Jeleń2

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznań, Poland; 2Institute of Food Technology of Plant Origin, Poznan University of Life Sciences, Poznań, Poland; 3Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznań, Polande-mail: Łukasz Chrzanowski <[email protected]>

We studied the effect of rhamnolipid biosurfactant on pop-ulations of hydrocarbon degrading bacterial consortium during diesel and biodiesel/diesel blends (B20) degradation under full aeration or limited aeration with nitrate as main electron acceptor. Rhamnolipids persistence to microbial co-utilization was evaluated starting from initial concentra-tion of 150 mg/L, that corresponded to its critical micelle concentration (CMC). Real time quantitative PCR was used to quantify 16S rRNA gene sequences representing dif-ferent species constituting bacterial consortium. Diesel/biodiesel blends (B20) but not diesel biodegradation rates were increased due to rhamnolipids supplementations. No influence was observed under anaerobic conditions.Rham-nolipids were completely utilized within 8–10 days under aerobic conditions while under anaerobic condition com-plete removal would likely occur within 27–35 days for cul-tures grown on B20 and diesel. Biosurfactant depletion was followed by an increase in gene copies of Achromobacter sp. and Citrobacter sp. while Comamonadaceae and Sphingobacterium sp. were suppressed by rhamnolipids for aerobic cultures grown with diesel and B20 respectively. Under anaerobic conditions, regardless of the fuel type, rhamnolipids caused notable reduction of Sphingobacterium sp. number.AcknowledgementsThis study was funded by Grant 32-194/11 DS-MK.

P15.31

Rhamnolipids increase the phytotoxicity of diesel oil towards four common plant species in a terrestrial environmentDamian Ambrożewicz1, Łukasz Ławniczak1, Alicja Szulc1, Roman Marecik2, Paweł Cyplik2, Joanna Wojtera3, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznań, Poland; 2Department of Biotechnology and Food Microbiology, University of Life Sciences in Poznań, Poznań, Poland; 3Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznań, Poznań, Polande-mail: Damian Ambrożewicz <[email protected]>

The studies focused on assessing the influence of rhamno-lipids on phytotoxicity in diesel oil-contaminated soil sam-ples. Seed germination and plant growth inhibition tests were carried out at different rhamnolipids concentrations (ranging from 0 to 1200 mg/kg of wet soil) for four ter-restrial plant species: alfalfa, sorghum, mustard and cuck-ooflower. The experiments were performed in soil samples with a different diesel oil content (ranging from 0 to 25 ml/kg of wet soil). It was observed that the sole presence of rhamnolipids may cause phytotoxicity at various levels, which is especially notable for sorghum (germination index decreased to 41%). The addition of rhamnolipids to diesel-oil contaminated samples contributed to a significant in-crease in phytotoxicity. The most toxic effect was observed after a rhamnolipid-supplemented diesel oil biodegradation trial, carried out with the use of a hydrocarbon-degrading bacterial consortium. The addition of rhamnolipids (600 mg/kg) resulted in a drastic decrease in seed germination for all studied plant species and an inhibition of microbial activity, which was measured by TTC tests. These findings indicate, that the presence of rhamnolipids may consider-ably increase the phytotoxicity of diesel oil, therefore their use at high concentrations during in situ bioremediation processes in a terrestrial environment should be avoided.AcknowledgementsThis study was funded by Grant 32-194/11 DS-MK.


P15.32

Rhizosphere as a tool to introduce a soil-isolated hydrocarbon-degrading bacterial consortium into a wetland environmentGrzegorz Framski1, Roman Marecik2, Łukasz Chrzanowski3, Paweł Cyplik2, Agnieszka K. Olejnik-Schmidt2, Łukasz Ławniczak3, Alicja Szulc3, Piotr Lisiecki3, Jacek Staniewski3

1Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland; 2Department of Biotechnology and Food Microbiology, University of Life Sciences in Poznań, Poznań, Poland; 3Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznań, Polande-mail: Piotr Lisiecki <[email protected]>

An attempt to incorporate a soil-isolated bacterial consor-tium capable of degrading hydrocarbons into the sweet flag rhizosphere was made in order to introduce the plant-bacteria combination into a wetland environment. The colonization of the rhizosphere occurred after a diesel oil degradation trial, where the plant was inoculated with the bacterial consortium. Both plant and bacterial growth were monitored. The PCR-RFLP method was used to verify the presence of the soil-isolates in the plants internal tissues and TTC-dehydrogenase measurements were employed to assess the microbial activity. The incorporation of bacterial cells into the sweet flag rhizosphere resulted in an increased diesel oil removal efficiency (by 50%) and enhanced bacte-rial and plant growth. Afterwards, the colonized sweet flag rhizomes were planted within a lake shoreline to evaluate the long-term prevalence of microorganisms in the natural environment. The bacterial DNA of the oil-degrading con-sortium species was detected in the harvested plants even after 8 months.AcknowledgementsThis study was funded by Grant 32-194/11 DS-MK.

P15.33

How biodegradable are the commercial ‘biodegradable bags’?Marta Woźniak1, Rafał Giszter1, Agnieszka Kutrowska2, Adrian Czaban2, Alicja Szulc1, Łukasz Ławniczak1, Joanna Wojtera2, Arkadiusz Kloziński1, Łukasz Chrzanowski1

1Institute of Chemical Technology and Engineering, Poznan University of Technology, Poznań, Poland; 2Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznań, Poznań, Polande-mail: Marta Woźniak <[email protected]>

A trend to replace polyethylene shopping bags with biode-gradable alternatives can be observed. However are these eco-bags as environmentally friendly as the producers claim or is it more of a sales pitch? With this in mind we inves-tigated the biodegradability of four commercially available market bags in the terrestrial environment. The first type of bags was made of polyethylene combined with starch, the second type contained degradation promoters, the third type was made of pure polyethylene and the fourth was a paper-bag. The studies included analyzing the changes among the mechanical properties of the material (elonga-tion, puncture and friction tests along with SEM pictures of the material surface), which occurred as a consequence of the microbial attack, and subsequent determination of the emitted CO2. The tests have been conducted after 3, 6, 9 and 12 months, accordingly. The obtained results indicate that only the paper-bags were readily biodegraded, while the bags containing polyethylene were resistant to micro-bial attacks. Although the mechanical properties of these bags deteriorated over time, this phenomenon had little to do with biodegradation.AcknowledgementsThis study was funded by Grant 32-194/11 DS-MK.


P15.34

Isolation of active substances from juice derived from cabbage for phytoremediation and biofumigation of soilsIrena Niksinska, Ryszard Heropolitanski, Edward Grzywa, Małgorzata Machałowska

Industrial Chemistry Research Institute, Departament of Polyester, Epoxides and Polyurethanes, Polande-mail: Niksińska <[email protected]>

It was found that the white cabbage is useful for a proc-ess of phytoremediation (soil purification from metals and harmful organic compounds) and biofumigation (soil pro-tection).Usually, the plants used for phytoremediation are considered as a waste, while cabbage accumulates heavy metals and a chemical stress associated with that stimulates the production of glucosinolates. These compounds, spe-cifically the products of their decomposition, known as isothiocyanates, have the ability to inhibit the growth of parasites, bacteria and fungi. Biocidal activity of isothiocy-anates can be compared with the effectiveness of synthetic pesticides. For isolation of impurites and active substances from a cabbage juice the method of sorption and desorp-tion of ionic and nonionic resins was used. The work was carred out in the following three steps: removal of heavy metals-isolation of organic pollutants-sorption and des-orption of active substances. The resulting concentrated solutions containing the active substances will be used to obtain the biopreparation. AcknowledgementsThe work is a part of the POIG projet: The use of white cabbage for phytoremediation and soil biofumigation”.

P15.35

Phytotoxicity of Diesel oil towards selected plant species and their response to petroleum hydrocarbons upon presence of petroleum-degrading microorganismsJoanna Wojtera1, Łukasz Chrzanowski2, Weronika Graj1, Elisa Pena Jimenez3, Piotr Kaczmarek1, Aneta Piechalak1, Arleta Małecka1, Barbara Tomaszewska1

1Biochemistry Department, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznań, Poznań, Poland; 2Institute of Chemical Technology and Engineering, Poznań University of Technology, Poznań, Poland; 3Facultad de Ciencias, Universidad de Granada, Spaine-mail: Wojtera <[email protected]>

Due to uncontrolled release from petroleum industry, ac-cidents during transportation and large-scale ecological catastrophes, the pollution by total petroleum hydrocar-bons (TPH) has been a huge threat to the natural envi-ronment and, in consequence, to human health. The mu-tualistic interaction between soil microbes and plants has been utilized in the RHIZOREMEDIATION technique that enables the uptake, inactivation or complete removal of organic xenobiotics from the polluted environment. In order to investigate the rhizoremediation potential, we have analyzed the phytotoxicity of Diesel oil towards Indian mustard, alfalfa, and two varieties of rapeseed – low and high erucic acid rapeseed (LEAR and HEAR, respectively). Additional germination tests were conducted upon inocu-lation of soil with petroleum-degrading microbial consor-tia (bioaugmentation). The changes in the seed germina-tion of the tested plants, their root elongation, fresh and dry biomass were determined upon all applied conditions. The initial results indicated that the exposition to Diesel oil inhibited the root elongation of all plants, but in case of LEAR and HEAR, low soil contamination with Diesel oil (3450 mg/kg) had a stimulating effect on their root growth. When considering fresh biomass of all investigated plant species at the increasing pollution, the greatest loss was ob-served in case of Indian mustard and LEAR. Interestingly, in the most contaminated soil (27.600 mg/kg), the fresh biomass of HEAR was 21% higher than in control. The most apparent increases in the dry biomass were observed for LEAR and HEAR (20% and 89%, respectively). Upon presence of petroleum-degraders in the polluted soil, only some from the tested bacteria stimulated the fresh and dry biomass increase of LEAR and HEAR varieties. In case of Indian mustard and alfalfa these parameters were reduced under the same conditions. Upon exposition to Diesel oil, the oxidative stress in the rapeseed shoots and roots is re-flected by an enhanced activity of catalase (CAT), as well as ascorbate peroxidase (APX). The impact of bacteria con-sortia on the oxidative stress level is observed only to some extend in rapeseed plants.AcknowledgementsThis project is supported by the Foundation for Polish Science, from the HOMING PLUS Programme, co-financed from EU structural funds un-der Action 1.2 ‘Strengthening the human resources potential of science’ of the Innovative Economy Operational Programme 2007–2013.


P15.36

Catalytic properties of yeast Saccharomyces cerevisiae for xanthohumol transformationAnna K. Żołnierczyk, Monika Stompor, Wanda Mączka, Mirosław Anioł

Wrocław University of Environmental and Life Science, Department of Chemistry, Wrocław, Polande-mail: Wanda Mączka <[email protected]>

Flavonoids are a group of about 7 000 chemicals common-ly found in plants. Their presence is high in fruit (espe-cially citrus), vegetables, pulses and many medicinal plants. Cheap and readily available source of flavonoids are spent hops — a waste product from extraction of hops, super-critical carbon dioxide. The main flavonoid from hops is xanthohumol — biologically active compound — is a po-tential anticancer agent, has antioxidant, antimutagenic, an-tiviral (including anti-HIV-1) activity (Stevens et al., 2004). It has also been patented as a cure for osteoporosis (US Patent 1997). Because of these properties xantohumol and its derivatives are used in cosmetic industry, pharmaceutical and food as a food additives. One way of obtaining new derivatives are biotransformation.Cheap, accessible and easy to use source of biocatalyst are baker’s yeast (Saccharomyces cerevisiae) (Csuk et al.,1991). They can be used as a living cell cultures, pressed, dry, lyophi-lized or immobilized. Due to the large amount of enzymes produced in yeast can catalyze a wide variety of reactions: oxidation and reduction, ester hydrolysis or formation of bonds between carbon atoms. The reactions catalyzed by yeast can be carried out in an aqueous, two-phase systems or in organic solvents. The biotransformations are com-petitive in relation to chemical methods in economic terms and also meet the environmental requirements - are an ex-ample of “green chemistry”.The aim of the experiment was to select the best reaction medium for the transformation of xanthohumol carried out by baker’s yeast, which were immobilized by a standard method in the alginate. Reactions were carried out for 24 hours at 28°C in distilled water, glycine buffer (pH 8.5), n-heptane, acetonitrile, ethyl acetate, acetone, tetrahydro-furan, and the ionic liquid [bmim][PF6]. The progress of reaction was studied using chromatographics methods: TLC and HPLC. The best results were achieved for the reaction conducted in an environment of tetrahydrofuran (24% conversion by HPLC).ReferencesCsuk R et al. (1991) Chem Rev 47-97.Stevens JF et al. (2004) Phytochemistry 65: 1317-1330.US Patent and Trademark Office, United States Patent Number: 5. 679. 716, 1997.

P15.37

Sequence diversity of the plasmid replication gene repC in the Rhizobium leguminosarum nodule populationPiotr Koper, Andrzej Mazur, Jerzy Wielbo, Grażyna Stasiak, Karol Czubak, Paweł Czubak, Kamil Żebracki, Anna Skorupska

University of Maria Curie Skłodowska in Lublin, Institute of Microbiology and Biotechnology, Department of Genetics and Microbiology, Polande-mail: Piotr Koper <[email protected]>

Rhizobia are soil bacteria, establish symbiosis with legumes, providing them with fixed nitrogen enabling their growth on nitrogen-limited soils. Rhizobial genomes comprise cir-cular chromosome and plasmids. The plasmid portfolio varies markedly in terms of sizes, numbers, and incompat-ibility groups. Maintenance of low-copy plasmids in rhizo-bial population requires active segregation and replication systems. Rhizobial extrachromosomal replicons are usu-ally equipped with repABC genes that control their replica-tion and partition. Rhizobial strains carry more than one repABC plasmid, which means that this plasmid family em-braces several incompatibility groups. Rhizobium leguminosarum bv. trifolii TA1 strain (RtTA1), has a single circular chromosome and four large plasmids, named pRleTA1a-d, each carrying repABC genes. The smallest plasmid, pRleTA1a is recognized as pSym, due to carrying nodulation — nitrogen fixation genes.The aim of this study was to analyze diversity of the plas-mid replication gene repC to infer evolutionary history of plasmids of Rhizobium leguminosarum (Rle) biovars, i.e., trifo-lii, viciae and phaseoli, inhabiting common soil environment. The group of 28 Rle strains isolated from nudules of clo-ver, vetch and pea grown in the same site in cultivated soil was selected. The selected strains differed significantly with respect to the plasmid number and size within and among biovars. In each strain, the repC genes were amplified and sequenced using primers based on respective sequences of RtTA1 plasmids. In the phylogenetic tree repC sequences obtained with the individual primer pairs clustered together forming separate clades containing repC of different bio-vars. The clusters may be correlated with different incom-patibility groups of the plasmids and may suggest com-mon origin of respective plasmids. Hybridizations with individual RtTA1 repC genes as molecular probes to PFGE separated replicons of examined strains indicated that par-ticular repC were located on plasmids, which significantly vary in size and plausibly in the genetic content between different strains.We concluded that extrachromosomal (or accessory) ge-nome of rhizobial population representing different bi-ovars of Rle, consist of a pool of widespread repABC plas-mids which may work in “common genomic framework” and of others plasmids equipped with diverse (repABC presumably) replication system. This further supports the hypothesis of pangenomic structure of rhizobial genomes.

environmental biotechnology lectures l15 - actabp.pl · environmental biotechnology lectures ......

Documents