Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S397

alpha-amylase.A yeast strain capable of producing an extracellular alpha-

amylase was isolated from environment. Sequence analysis of theD1/D2 domain of the 26S rDNA gene was used to identify the iso-lated yeast, revealing a 93.5% similarity with Candida ontarionensis.However, it is not possible to assign a genus and species designationdue to the small similarity with reported species.

Several basal culture media supplemented with soluble starchwere tested for the production of extracellular alpha-amylase.Castaneda-Agullóıs liquid basal medium gave the highest enzymeproduction, so that it was used in further studies.The effect of dif-ferent carbon and energy sources (glucose, maltose, soluble starchand glycerol) on enzyme production was evaluated in aerobic batchcultures. Moreover, aerobic chemostat cultures of the isolated yeaststrain were carried out under starch-limited conditions. Concen-trations of biomass, glucose, reducing and total sugars, as well asenzyme activity were measured.

The highest enzyme production was obtained when the yeastwas grown in batch cultures with soluble starch as the carbon andenergy source. Enzyme production reached its maximum level at25 h of incubation; thereafter enzyme activity remained almostconstant during stationary growth phase. Enzyme production washigher in a mechanically-stirred tank bioreactor than in agitatedflasks.

Results from batch experiments carried out with different car-bon sources suggest that the enzyme is inducible and undercarbon-catabolite repression.

Chemostat studies showed that the highest enzyme productionwas obtained at dilution rates higher than 0.07 h−1. Dextrin, glucoseand maltose were identified as the main enzyme reaction products.

doi:10.1016/j.jbiotec.2010.09.513

[P-I.148]

Effect of Acid Tolerance Response on Recombinant Streptoki-nase Production in Fed-batch Cultures of Lactococcus lactis P170Expression System

Karthikeyan Thillai ∗, Guhan Jayaraman

Indian Institute of Technology-Madras, IndiaKeywords: Lactococcus lactis; Acid tolerance response; Fed-batch;Streptokinase

Recombinant protein production in Lactococcus lactis fermen-tation culture is mainly affected by the acidic end products.Accumulation of lactic acid under anaerobic condition leads to acidstress which inhibits both cell growth and recombinant proteinproduction. Acid Tolerance Response (ATR) cannot be completelysuppressed by adding neutralizing agent at controlled pH cul-tures. Development of Acid Tolerance Response depends on bothpH and medium composition. In this study, the effect of acid tol-erance response on recombinant streptokinase expression wasinvestigated in fed-batch cultures of Lactococcus lactis P170 expres-sion system. This expression system is based on an autoinducibleP170 promoter and induced by low pH (upregulated at pH below6.5) in the transition to stationary phase. However, the physi-ological characterization of the promoter showed that the realinducer of P170 was the lactate concentration. The P170 Expres-sion System is induced when a certain threshold of lactate isreached in the culture. Thus, the lactate concentration controlspromoter activity and acid tolerance response. The optimal lev-els of lactate for the production of recombinant streptokinase atpH 6.5 were determined. Fed-batch cultures of recombinant Lacto-coccus lactis P170 expression system for producing streptokinase

were performed at different specific growth rates before induc-tion and at a constant value after induction by the method ofpseudo-exponential feeding. Experimental results showed that therecombinant streptokinase production could be improved by sup-pressing acid tolerance response and more than 60 fold increase inrecombinant streptokinase expression has been achieved. Also, sys-tematic approach to evaluate the effect of acid tolerance responseon recombinant streptokinase expression was carried out by addinglactate into culture media at different cell growth phases. It wasalso found that the lactate inhibition on cell growth occurred in thebatch growth phase while its inducer role on recombinant strep-tokinase expression in the post-induction phase.

doi:10.1016/j.jbiotec.2010.09.514

[P-I.149]

Anaerobic acidogenic digestion of a dephenolized olive millwastewater in a biofilm reactor packed with ceramic filters

L. Bertin 1,∗, A. Scoma 1, C. Bettini 1, L. Marchetti 1,2, F. Fava 1

1 Department of Civil, Environmental and Material Engineering(DICAM), University of Bologna, Italy, Italy2 Interuniversitary Consortium “Chemistry for the Environ-ment”(INCA), Italy, ItalyKeywords: Olive mill wastewater; Polyphenols recovery; Packedbed biofilm reactor; Acidogenic fermentation

The exploitation of organic waste in the biotechnological pro-duction of biopolymers such as polyhydroxyalkanoates is of greatinterest in the perspective of reducing their costs. To this aim, athree-stage integrated anaerobic-aerobic process fed with olivemill wastewaters (OMWs) was proposed (Dionisi et al., 2005). Inits first step, the waste is digested under acidogenic conditions inorder to obtain a volatile fatty acids (VFAs) enriched effluent tobe fed to the PAH producing reactor. OMW polyphenols, whichadversely affect the waste fermentation, are natural antioxidantswhose exploitation can concern several industrial fields such ascosmetic and food industry. Thus, an integrated chemical-biologicalprocess for OMW polyphenols recovery by liquid-solid extractionand for the continuous acidogenic fermentation of the depheno-lized waste was developed.

The employed OMW (pH = 4.5) had a total phenol content of4.6 g/L, while COD and VFA concentrations were 55 and 8.4 gCOD /L,respectively. As a result of the adsorption pre-treatment, carriedout with resin Amberlite XAD16 as the solid phase (0.7 g/L, con-tact time = 2 hours), the 90% of OMW polyphenols were removedtogether with the 25% of the COD, while VFA concentration and pHdid not vary appreciably. The resulting wastewater was processedin a biofilm reactor packed with ceramic filters

developed in a recent study (Beccari et al., 2009). On the basisof the results obtained from a preliminary batch experiment, thepH of the influent flow was correct to 7.0 and the reactor wasthermostated at 25 ◦C and fed with an OLR of about 6 gCOD/L/day.The process effluent, whose pH was 6.2, had a total VFA and CODconcentrations of 19.02 and 25.94 gCOD/L, respectively. A signifi-cant enhancement in terms of VFA production yield with respectto the former experience (Beccari et al., 2009) was achieved. Inparticular, butyric and acetic acids were the main detected VFAsin the obtained effluent where they represented the 28 and 27%,respectively, of the whole VFA mixture. The reactor biofilm is undercharacterization by means of molecular biology approaches.

S398 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

References

Beccari, M., et al., 2009. J Chem Technol Biotechnol 84, 901–908.Dionisi, D., et al., 2005. Wat Res 39, 2076–2084.

doi:10.1016/j.jbiotec.2010.09.515

[P-I.150]

Evaluation of the Recombinant E. coli BL21 Strains ProducingProchymosin in the Biotechnological Processes

Leila Badiefar ∗, Mahvash Khodabande, Gholamreza Ahmadian

National Institute of Genetic Engineering and Biotechnology, IslamicRepublic of IranKeywords: Prochymosin; Protein expression; Sulobilization;Refolding

The levels of protein expression and the amount of cell bulkare some of the very important parameters in the biotechnologicalprocesses involving high-yield production. Chymosin is an aspartylproteinase that is found in the abomasums of unweaned calves.In this research, we compared protein expression levels, total cellmass and production efficiency at two recombinant E. coli BL21 car-rying the complete and exon-6 less prochymosin under flask andfermenter culture conditions. Studied variables were IPTG concen-tration, OD for induction of protein production, time after inductionand temperature. The best growth conditions was selected in therange of variables under experiment. The level of prochymosin pro-duction and the final OD were measured at mentioned conditions.Also, the production efficiencies of the two prochymosin formswere evaluated. The level of prochymosin production was thencompared by fermentation of two recombinant E. coli BL21 strainsunder the optimized conditions. The results indicate that the levelsof protein expression and final OD under optimum conditions are33.53 and 5.88 for E. coli BL21 harboring pET- prochymosin and 45.4and 7.82 for E. coli BL21 harboring pET- exon 6less prochymosin,thus their production efficincies will be 197.15 and 355.02 respec-tively. Whereas every two types of prochymosin are producedas insuloble inclusion bodies in the cytoplasmic space, sulobi-lization and refolding of these proteins are necessary. We couldsolubilize and refold complete and shortened prochymosin withefficiency of 47 and 56% respectively. Therefore the results relatedto the efficiencies of the prochymosin production, solubilizationand refolding of inclusion bodies indicate that use of E. coli BL21harboring pET- exon 6less prochymosin is very better than E. coliBL21 harboring pET- prochymosin at biotechnological process.

doi:10.1016/j.jbiotec.2010.09.516

[P-I.151]

Separation of p-aminobenzoic acid using liquid membrane inpresence of phase modifier

L. Kloetzer 1, A.C. Blaga 1,∗, A.I. Galaction 2, D. Cascaval 1

1 Technical University of Iasi, Romania2 University of Medicine and Pharmacy of Iasi, RomaniaKeywords: p-aminobenzoic acid; Pertraction; Liquid membrane;Phase modifier

The studies on facilitated pertraction with Amberlite LA-2 ofPABA in presence of 1-octanol added into the membrane phaseindicated the significant increase of the acid initial and final massflows. Compared with the facilitated pertraction without alcohol,

Fig. 1. Influence of pHS on factors FN and FP (pHF = 4, Amberlite LA-2 concentra-tion = 40 g/l, rotation speed = 500 rpm; 1-octanol concentration = 10% vol.).

the acid mass flows have been amplified up to 3 times, but themagnitude of this effect has to be correlated with the pH-gradientbetween the feed and stripping phase, carrier and 1-octanol con-centrations inside the liquid membrane and mixing intensity. Fig. 1.

Contrary to this positive effect, the addition of 1-octanol inducedthe diminution of the permeability factor, because the extractionrate of PABA into the membrane phase exceeded that of reextrac-tion from this phase, the transport capacity of the investigatedpertraction system being limited. For this reason, the aim of thefuture studies is to optimize the reextraction process of the acidfrom the liquid membrane to the stripping phase.

doi:10.1016/j.jbiotec.2010.09.517

[P-I.152]

Bioprocess systems engineering applied to a multipurpose sec-ond generation ethanol-from-sugarcane plant: assessing theperformance of local and global optimization algorithms

F.F. Furlan 1,4, R.C. Giordano 1,4,∗, A.J.G. Cruz 1,4, C.B.B. Costa 1,4,A.R. Secchi 2,4, R.P. Soares 3,4

1 Federal University of Sao Carlos, Brazil2 COPPE, Federal University of Rio de Janeiro, Brazil3 Federal University of Rio Grande do Sul, Brazil4 Chemtech, BrazilKeywords: Biorefinery optimization; Second generationbioethanol; Sugarcane bagasse enzymatic hydrolysis; BioprocessSystems Engineering

Production of biofuels is presently at a technological crossroad. Ahard competition among technologies is in course and the winnerswill be defined by a combination of economical criteria, complianceto environmental restrictions, and process robustness.

Optimization of this complex and integrated industrial processmust aim at different targets: cost reduction, sustainability (use ofland, negative CO2 balance), effluent emissions, and water usagecutback. In this scenario, fine-tuned processes, operating at (near-)optimum conditions will have significant competitive advantage,and the application of Bioprocess Systems Engineering tools will beessential for this task.

This work assesses the performance of two algorithmsfor defining optimal operation points of a second generationbioethanol-from-sugarcane plant. A highly energy-integrated pro-cess, using surplus bagasse and lignin for steam generation, is usedas case study. First and second generation anhydrous and hydrated