S568 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

Enhancing oil degradation in soil by stimulating endogenousmicrobial activity

Maria Teresa Dell’Abate 1, Stefano Mocali 1,∗, Laura Bardi 2, LetiziaPompili 3, Anna Benedetti 1

1 CRA – Centro di Ricerca per lo studio delle Relazioni tra Pianta e Suolo,via della Navicella 2/4 – 00184 Roma (Italy)2 CRA - Centro di Ricerca per lo studio delle Relazioni tra Pianta e Suolo,sede operativa periferica di Torino (Italy)3 CRA – Sede Centrale, via Nazionale 82 - 00184 Roma (Italy)Keywords: bioremediation; petroleum; microbial diversity; soilpollution; compost

E-mail address: stefano.mocali@entecra.it (S. Mocali).

Soil and groundwater contamination with oil compounds isa relevant environmental issue. Although bioremediation of oil-polluted soils can be achieved by using consortia of oil-degradingbacteria as well as endogenous soil microorganisms, one of the mostlimiting factors to effective and complete degradation of hydrocar-bons is their scarce bioavailability to soil microorganisms mainlydue to limited solubility in aqueous media. In this study the effectsof the addition of cyclodextrin (Cy), compost (C) and a combina-tion of them (Cy+C) on the bioremediation of oil-polluted soil werecompared in a mesocosm-scale experiment. Soils added with threedifferent amounts of oil (0,1%, 2% and 6%) were incubated during190 days.

The level of hydrocarbons was monitored through gas-chromatography (GC) whereas microbial biomass (Cmic) and soilrespiration (C0) were determined to assess the microbial responseto both the pollution and the treatments. Functional and geneticdiversity of soil microbial communities were analyzed by commu-nity level physiological profile (CLPP) and Denaturing Gradient GelElectrophoresis (DGGE), respectively.

The results showed a significant reduction of hydrocarbons con-tent in soils with 0.1% and 2% oil when Cy or Cy+C were added,whereas in soils with the highest pollution (6% oil) no effectsinduced by any treatment were detected. In contrast the addition ofC increased Cmic and C0 values but appeared to be ineffective in theremediation process. DGGE suggested that the addition of hydro-carbons selected for different bacterial communities more than thetreatments. CLPP indicated that microbial metabolism decreasedin higher-polluted soils, but the addition of Cy increased microbialcatabolic profiles more than C or Cy+C.

In conclusion the addition of Cy appeared to stimulate the biore-mediation process by increasing the bioavailability of hydrocarbonswhereas the addition of compost seemed to stimulate the growth ofendogenous microorganisms but appeared to poorly enhance theremediation process.

doi:10.1016/j.jbiotec.2010.10.042

Estimation of antibiotic resistance of microorganisms with helpthe electro optical analysis of microbial cells

Olga I. Guliy ∗, Oleg V. Ignatov

Institute of Biochemistry and Physiology of Plants and MicroorganismsRAS, 13 Entuziastov av., Saratov 410049, Russia

Study of the adaptation of microbes to antibiotic action is animportant problem that is of theoretical and applied significance.

We examined the effect of ampicillin and kanamycin on the elec-trophysical characteristics of ampicillin- and kanamycin-sensitiveand ampicillin- and kanamycin-resistant Escherichia coli cells.

Ampicillin is a �-lactamic antibiotic, it is a broad-spectrumagent that is active specifically toward E. coli. One can assumethat changes in cell morphology and cell-wall disturbances inantibiotic-resistant microorganisms should lead to changes in theirelectrophysical characteristics. These changes are reflected in alter-ations in the electro-optical (EO) characteristics of cell suspensions,which are recorded during experiments using electric-field cell ori-entation.

Kanamycin, an inhibitor of protein synthesis, is an aminogly-caside antibiotic of the oligosaccharide family. This antibiotic waschosen because, in contrast to other bacteriostatic antibiotics thatinhibit protein synthesis, it is weakly bactericidal. It has enteredmicrobial cells through porin channels, kanamycin suppresses pro-tein synthesis and does not cause changes in cellular morphologicalcharacteristics.

Substantial changes in the orientational spectra (OS) of suspen-sions of cells incubated with various ampicillin and kanamycinconcentrations took place only at the first five frequencies of theorienting electric field (10–1000 kHz).

The maximal change in the magnitude of the electro-optical sig-nal occurred at 50 �g/ml of ampicillin and 10 �g/ml of kanamycin.Under the action of ampicillin and kanamycin, sensitive and resis-tant E. coli strains gave different EO effects. It follows that thesensitive and resistant E. coli strains exhibit different of theEO effect on the action of ampicillin and kanamycin. Thus, thesuspension-OS changes occurring under the effect of ampicillin andkanamycin may be used as a test for resistance to this antibiotic inthe cells studied.

This work has benefited from grants of RFBR №09-02-12442-ofi m.

doi:10.1016/j.jbiotec.2010.10.043

Electrophysical analysis of microbial cells for bioanalyticalapplications

O.V. Ignatov 1,∗, O.I. Guliy 1, V.D. Bunin 2, V.V. Ignatov 1

1 Institute of Biochemistry and Physiology of Plants and Microorgan-isms, Russian Academy of Sciences, 13 Pr. Entuziastov, 410049 SaratovRussia2 EloSystem GbR, Berlin, Germany

Mmeasurement of electrophysical properties of microbial cellsmay be used for creation of new kind of biosensor systems. Therewere investigated the microbial cells of some strains with pre-liminary metabolic enzyme system of toxic compounds. Theseprocesses conduce to the redistribution of the charges in the micro-bial cells and may be register by electro-optical (EO) methods. EOanalysis is based on the recording of changes in optical character-istics of cell suspensions under the orienting effect of an electricfield. Redistribution of the charges in the microbial cells may beused for determination of substrates of enzyme reaction and finallyfor determination of substrate concentration.

Same approach may be used for studies of microbial cells andsome biological agents (antibodies and phages) binding. Since theAC electrokinetic effects depend on dielectric properties of biopar-ticles, their composition, morphology, phenotype, the medium, andthe frequency of applied electrical field, the EO properties of cellsuspensions were used for discrimination of bacteria before andafter selective binding with antibodies.

There were shown the determination of the presence of partic-ular bacteria within a mixed sample may be achieved by selectionand matching of antibodies specific to individual bacterium typesand by comparing spectra of bacterium in the presence and inthe absence of specific antibody. Same principles were used for

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S569

investigations of bacteria – phage interaction. Integration of theelectro-optical approach with a bioselective binding agents has thefollowing advantages: 1) bacteria from biological samples need notbe purified and 2) exogenous substrates and mediators are notrequired for detection.

This work has benefited from grants of RFBR №09-02-12442-ofi m.

doi:10.1016/j.jbiotec.2010.10.044

Estimation of kinetic parameters of a lactic acid production pro-cess from sugar cane molasses using genetic algorithm

B.H. Lunelli 1,∗, D.I.P. Atala 2, M.R. Wolf Maciel 1, R. Maciel Filho 1

1 Laboratory of Optimization, Design and Advanced Control, Schoolof Chemical Engineering, State University of Campinas, Campinas, SP,Brazil2 Sugar Cane Technology Center, Piracicaba, SP, Brazil

The objective of this work is to estimate the parameters set of thekinetic model of lactic acid production from sugarcane molasses.The model was developed in FORTRAN language and describes thelactic acid production, sucrose consumption, cell growth and con-siders the inhibitory effect of substrate and products. The kineticparameters were determined using substrate, product and biomassconcentration obtained in a batch fermentative process. Fermen-tation was carried out in a bioreactor (3 L) with 2.5 L workingvolume. Sugarcane molasses without pre-treatment was dilutedwith distilled water to obtained sucrose initial concentration of 12g/L approximately. The fermentation medium was enriched with5 g/L of yeast extract. The temperature was maintained at 340C,the agitation speed at 200 rpm and pH at 5.0 by adding 4MNaOH.The fermentation was performed under anaerobic conditions. TheLabVIEW® software was used for monitoring and controlling pro-cess parameters during fermentation. To estimate the parameterswas used genetic algorithm (GA). The genetic algorithm is an effec-tive stochastic global search and is based on the genetics andnatural evolution principles of the species. The mechanism of theGA technique occurs with successive modifications of the indi-viduals or chromosomes of population through the application ofselection, crossover, and mutation operators. The results shownthat the GA technique it is adequate to fit of the kinetics param-eters the lactic acid production model, and describes satisfactorilythe fermentative process developed.

doi:10.1016/j.jbiotec.2010.10.045

Ethyl lactate production using reactive distillation: experimen-tal and simulation

B.H. Lunelli 1,∗, D.I.P. Atala 2, N. Oliveira 2, M.R. Wolf Maciel 1, R.Maciel Filho 1

1 Laboratory of Optimization, Design and Advanced Control, Schoolof Chemical Engineering, State University of Campinas, Campinas, SP,Brazil2 Sugar Cane Technology Center, Piracicaba, SP, Brazil

Ethyl lactate is an important organic ester, which is biodegrad-able and can be used as food additive, perfumery, flavor chemicalsand can effectively replace oil-based solvents. The esterification oflactic acid with ethanol can be used as a method to obtain highlypurified lactic acid from fermentation broths. In the present work,ethyl lactate was produced from aqueous lactic acid solution andethanol in a continuous reactive distillation process in the presenceof heterogeneous catalyst (cationic exchange resin) and homoge-

neous catalyst (sulphuric acid) with aim to verify the feasibility ofthe ethyl lactate production from renewable resources. Lactic acidused in the process was produced by fermentative process usingsugarcane molasses and bacterial strains isolated from industrialalcoholic fermentation. Factorial experimental design was used toidentify the influence of different operating parameters such asreaction temperature, ethanol/lactic acid feed molar ration and theeffect of the catalyst type in the process. The ethyl lactate concen-tration was analyzed by gas chromatography (GC) and the bestresult was obtained when ethanol and lactic acid were feed in a1.3:1 molar ratio. A model was proposed, and the simulation of thereactive distillation process to ethyl lactate production was carriedout using Aspen simulator.

doi:10.1016/j.jbiotec.2010.10.046

Observed kinetics of the continuous growth and ethanol pro-duction of the genetically engineered yeast BHL01

D.L. Yin, Q. Li, F. Li, X.M. Ge, X.Q. Zhao, F.W. Bai ∗

Department of Bioscience and Bioengineering, Dalian University ofTechnology, Dalian, China

E-mail address: fwbai@dlut.edu.cn (F.W. Bai).

When cells self-flocculated and form flocs with suitable sizedistribution, they can be immobilized within bioreactors, withoutconsumption of supporting materials. Theoretically and econom-ically, this kind of self-immobilized cells are superior to cellsimmobilized with various supporting materials, making themmore suitable for continuous production of bulk commodities likeethanol, which, as a typical primary metabolite, its production istightly associated with yeast cell growth that could be seriouslycompromised by the physical constraint of supporting materials,and on the other hand, its market price is too low to accommodatethose extra costs associated with the preparation of immobilizedyeast cells at large scale and consumption of supporting materialsas well.

Kinetics is the prerequisite for process design and optimization.However, yeast flocs, which are much larger than regular yeastcells, present different kinetic behaviours characterized by moder-ate mass transfer limitation (Ge et al., 2006). In this report, observedkinetic behaviours of the genetically engineered self-flocculatingyeast BHL01 were studied under continuous fermentation condi-tions. Media with different glucose concentrations were fed intothe fermentation system at different dilution rates. The size dis-tribution of the yeast flocs was monitored online by the focusedbeam reflectance measurement (FBRM) system (Ge et al., 2005),and its impact on the kinetic behaviours of the yeast flocs was thusstudied. By incorporating the effectiveness factor into the intrinsickinetic equations, the observed kinetic models were establishedfor the continuous ethanol fermentation with this engineeredself-flocculating yeast strain, which can be used to simulate andoptimize the corresponding fermentation process.

Reference

Ge, X.M., Zhang, L., Bai, F.W., 2006. Impact of the floc size distributions on observedsubstrate uptake and product formation rates. Enzyme Microbial Technol 39,289–295.

Ge, X.M., Zhao, X.Q., Bai, F.W., 2005. Online monitoring and characterization of floc-culating yeast cell flocs during continuous ethanol fermentation. BiotechnolBioeng 90, 523–531.

doi:10.1016/j.jbiotec.2010.10.047