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.