Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S511

lus formation was obtained. It was possible in vitro propagation R.Crispa from seeds, but it’s response to growth regulators evaluatedwas poor and no plants were regenerated

doi:10.1016/j.jbiotec.2010.09.806

[P-P&F.104]

Efficient Heterotrophic Culture of Chlorella USTB-01 by Feedingthe Mixture of Glucose and A Nitrogen Containing Compound

Q.Q. Xu, S. Yang, Z.J. Wang, Z. Yan, X. Jia, H. Yan ∗

University of Science and Technology Beijing, ChinaKeywords: Chlorella USTB-01; Fed-batch culture; Glucose; Nitro-gen source

The heterotrophic culture of Chlorella USTB-01 was investigatedin this paper. Initial above glucose of 20 g L−1 or potassium nitrateof 4.0 g L−1 might apparently inhibit the growth of Chlorella USTB-01 in batch culture. When glucose and potassium nitrate fed weregradually increased with the augment of microalgal biomass infed batch culture, the specific growth rate of this microalga was0.079 h−1, which was much higher than 0.064 h−1 of one–time feed-ing. Under optimized feeding conditions, dry weight concentration(DWC) of microalgal cells at 48 h reached 45 g L−1, a level whichwas not previously reported in such a short period of time. Com-pared with nitrate and urea, ammonium supported the more rapidgrowth of this microalga and DWC of 52 g/L was obtained at 48 h,but the content of protein in Chlorella USTB-01 was the lowest,which indicated that the protein biosynthesis of Chlorella USTB-01 might be heavily postponed with the rapid growth. This study isvery important to help the heterotrophic industrial production ofChlorella.

doi:10.1016/j.jbiotec.2010.09.807

[P-S.1]

Systems Biology for Biotechnological Innovation

Fermentative production of L-lactic acid from cassava flourwastewater by Lactobacillus rhamnosus RL-1

CJB de Lima ∗, LF Coelho, J Contiero

Department of Biochemistry and Microbiology, Institute of BiologicalSciences, São Paulo State University, BrazilKeywords: cassava wastewater; fermentation; L-lactic acid; Lacto-bacillus rhamnosus

Advances in industrial biotechnology offer potential opportu-nities for economic utilization of agro-industrial residues such ascassava flour wastewater. Due to its rich organic nature, it can serveas an ideal substrate for microbial processes for the production ofvalue added products. Two cassava flour wastewater preparations(natural and hydrolyzed) were tested as culture media for l-lacticacid production by Lactobacillus rhamnosus RL-1. The microorgan-ism was able to grow and produce l-lactic acid in both preparations.The best result, 35.54 g l−1 lactic acid using 100% reducing sugar assubstrate after 48 h at 37 ◦C was obtained during the pH-controlledfermentation of hydrolysate medium. The volumetric productivityat the end of the fermentation was 0.656 g l−1 h−1, with a maximumof 1.31 g l−1 h−1 in the first 24 h. The volumetric productivity at theend of this fermentation was 0.59 g l−1 h−1, with a maximum of

0.82 g l−1 h−1 during the first 12 h. In addition, lactic acid producedfrom cassava flour wastewater by Lactobacillus rhamnosus sp. LR-1was almost optically pure l-lactic acid (over 99% of total lactic acidproduced).

doi:10.1016/j.jbiotec.2010.09.808

[P-S.2]

Medium composition and optimization of Lactic acid produc-tion by Lactobacillus plantarum Lmism-6 grown in molasses

L.F. Coelho ∗, C.J.B. de Lima, M.B. Piassi, J. Contiero

Department of Biochemistry and Microbiology, Institute of BiologicalSciences, São Paulo State University, Rio Claro, SP, Brazil, BrazilKeywords: Plackett-Burman; Lactobacillus plantarum; lactic acid;medium optimization

Lactic acid has been used in the pharmaceutical, textile, tanningand food industries as well as in the synthesis of biodegrad-able plastics. The aim of the present study was to optimize lacticacid production by Lactobacillus plantarum Lmism-6 growing inmolasses. A Plackett-Burman experimental design was used toevaluate seven medium components added to molasses (cornsteep liquor, sodium acetate, magnesium sulfate, manganese sul-fate, ammonium citrate, potassium phosphate and Tween 80).The corn steep liquor, K2HPO4 and Tween 80 increased lacticacid production. The concentrations of these three components aswell as the molasses were further optimized using the responsesurface method. A second-order polynomial regression model esti-mated that maximal lactic acid production of 91.61 g l−1 would beobtained when the optimal molasses, CSL, K2HPO4 and Tween 80values were 193.50 g l−1, 37.50 ml l−1, 2.65 g l−1 and 0.83 ml l−1,respectively. For the validation of the optimization of the mediumcomposition, studies were carried out using the optimized condi-tion to confirm the results of the response surface analysis and after48 h the lactic acid production using the shake-flask method was94.8 g l−1.

doi:10.1016/j.jbiotec.2010.09.809

[P-S.3]

Possible mechanism of flocculation and deflocculation ofunusual flocculent yeast

Hiroshi Nishihara ∗, Kenta Anzai, Masataka Niimura

Kagawa University, JapanKeywords: flocculation; deflocculation; yeast

Usual flocculent ability of brewer’s yeasts increases with grow-ing time. Floc-forming ability of cells of Saccharomyces bayanusIFO 1948, however, was the strongest 24 h after inoculation andthen decreased. The cells became almost dispersed 120 h afterinoculation. The present study was done to study mechanism offlocculation and deflocculation of the unusual flocculent yeast S.bayanus IFO 1948.

Cultivation was carried out at 30 ◦C with shaking. Yeast cellsgrown for 24 h and 120 h were harvested, washed with deionizedwater and then subject to enzyme-treatment or chemical modi-fication of the cell surface protein and carbohydrate components.Flocculent and co-flocculent ability was estimated as the degree offlocculation and co-flocculation (D.F. value)1).

Floc-forming ability of flocculent 24h-cells was destroyed bytreatment with proteolytic enzymes and chemical modification of

S512 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

cell surface protein and carbohydrate components. Significant co-flocculation occurred when 24h-cells deflocculated by chemicalmodification of cell surface protein component and the 24h-cellsdeflocculated by sodium periodate-oxidation were mixed. Thesefindings suggest that both cell surface protein and carbohydratecomponents play important parts in the intercellular interac-tion of the flocculent 24h-cells. Strong co-flocculation occurredwhen non-flocculent 120h-cells and flocculent 24h-cells weremixed. Treatment with proteolytic enzymes and chemical mod-ification of cell surface protein of non-flocculent 120h-cells didnot affect co-flocculation with flocculent 24h-cells but sodiumperiodate-oxidation deprived 120h-cells of co-flocculent abilitywith 24h-cells. The 24h-cells deflocculated by treatment with pro-teolytic enzymes or chemical modification of cell surface proteinfailed to co-flocculate with untreated non-flocculent 120h-cellswhile the 24h-cells deflocculated by sodium periodate-oxidationco-flocculated with untreated non-flocculent 120h-cells. Addi-tion of cycloheximide into growing culture 24 h after inoculationdepressed the decrease of floc-forming ability. These finding sug-gest that 24h-cells flocculate by interaction between cell surfaceprotein and carbohydrate (mannan) and 120h-cells fail to flocculatebecause of removal of protein component essential for flocculation.

1) J. Inst. Brew., 108, 187-192 (2002).

doi:10.1016/j.jbiotec.2010.09.810

[P-S.4]

Signalling Pathways and Cellular Effects Activated in Endothe-lial Cells by Exogenous AIMP1

V.C. Jackson ∗, S. Dewilde, A. Giuliano Albo, D. Corpillo, K. Lis, B.Canepa

LIMA, BioIndustry Park del Canavese S.p.A., Colleretto Giacosa (TO),ItalyKeywords: AIMP1, Angiogenesis, Endothelial cells, Signalling path-way, Phosphorylation, phosphoproteomics, VEGF, Receptor

AIMP1 (ARS-interacting multifunctional protein 1) is a cofactorof the Aminoacyl-tRNA synthetase complex, but also the precur-sor of EMAP II (Endothelial Monocyte-Activating Polypeptide II), socalled for its ability to act on endothelial cells and on many immunesystem cells (Quevillon et al., 1997). In particular stress conditions,both AIMP1 and EMAP II are found in the extracellular environ-ment, where they show cytokine properties, acting on many celltypes including endothelial cells, immune cells and fibroblasts (VanHorssen et al., 2006; Kao et al., 1992; Berger et al., 2000).

We studied the mechanisms of action of AIMP1 when releasedin the extracellular environment, focusing our attention on signalpathways activated in Porcine Aortic Endothelial Cells by exoge-nous AIMP1.

To do this, different techniques were employed includingbiological assays, immunofluorescence technique to study theentry of exogenous AIMP1 and its subcellular localization, a 2DE-phosphoproteomic analysis to reveal the signalling pathwaysresponsible for the cellular effects and qPCR to analyse transcrip-tional regulated genes.

AIMP1 has a dose-dependent role in angiogenesis, inhibitingECs proliferation at high concentrations and stimulating it at lowconcentrations (Park et al., 2002).

Immunofluorescence studies showed that exogenous AIMP1 isinternalized in ECs and accumulates in clots at the cell membranelevel.

AIMP1 activates ERK and JNK, but not AKT, through phosphory-lation after 10 minutes of treatment compared with a control. Using

a 2DE-phosphoproteomic approach, we confirmed the phosphory-lation of ERK upon AIMP1 treatment and identified several otherputative signalling molecules involved.

Preliminary results of qPCRs performed on a panel of genesfollowing ECs treatment with exogenous AIMP1 showed downreg-ulation of VEGF-A and VEGFR2.

In conclusion, treatment of ECs with exogenous AIMP1 leads toits cell entry, rapid phosphorylation of ERK and JNK and transcrip-tional regulation of the members of the VEGF pathway.

We hypothize that AIMP1 acts on PAE cells through a mem-brane receptor and we are currently trying to identify interactingmembrane proteins through affinity purification.

References

Quevillon, S., Agou, F., Robinson, J.C., Mirande, M., 1997. The p43 component ofthe mammalian multi-synthetase complex is likely to be the precursor ofthe endothelial monocyte-activating polypeptide II cytokine. J Biol Chem 272,32573–32579.

Van Horssen, R., Eggermont, A.M.M., ten Hagen, T.L.M., 2006. Endothelial monocyte-activating polypeptide-II and its functions in (patho)physiological processes.Cyt. Gr. Fact. Rev. 17, 330–348.

Kao, J., Ryan, J., Brett, G., Chen, J., Shen, H., Fan, Y.G., et al., 1992. Endothelialmonocyte-activating polypeptide II. A novel tumor-derived polypeptide thatactivates host-response mechanisms. J. Biol. Chem. 267, 20239–20247.

Berger, A.C., Tang, G., Alexander, H.R., Libutti, S.K., 2000. Endothelial monocyte-activating polypeptide II, a tumor-derived cytokine that plays an important rolein inflammation, apoptosis, and angiogenesis. J Immunother 23, 519–527.

Park, S.G., Kang, Y.S., Ahn, Y.H., Lee, S.H., Kim, K.R., Kim, K.W., et al., 2002. Dose-dependent biphasic activity of tRNA synthetase-associating factor, p43, inangiogenesis. J. Biol. Chem. 277, 45243–45248.

doi:10.1016/j.jbiotec.2010.09.811

[P-S.5]

Benchmarking Spain’s Biotechnology: a comparative perspec-tive

Isidre March-Chorda 1,∗, Jorge Niosi 2, Rosa M. Yagüe-Perales 1

1 University of Valencia, Spain2 Université de Québec à Montréal, CanadaKeywords: Spain; Venture; Patenting; Spin-off

Purpose of this study is to ascertain how different or similar isthe national system of Spanish biotechnology compared to those ofthe leading countries such as Canada, the United Kingdom or theUnited States.

The study is based on both public figures and a databasepurposely built on Spanish dedicated biotechnology firms. The sta-tistical method applied is the ANOVA analysis.

Main findings and results include:Older and larger companies linked with larger domestic groups

(mainly food, pharmaceutical or chemical) request patents mostoften.

Venture capital in Spain has eluded biotechnology and the lastfigures for 2008 and 2009 reveal a cut in the funding of biotechstart-ups.

In Spain, university spin-offs are younger than the average com-pany, and less likely to have venture capital and patents. They arenot particularly aiming at human health products, and are moreoften located in non core regions.

A massive 80% of the growing companies are older than theaverage, and 65% of them are research spin-offs. Growing firmstend to concentrate in a few larger metropolitan areas, while theirPatenting activity is not more intense than the average company.

Growing firms are more often owned by, or affiliated with, agroup of companies. Companies owned by individuals are almostalways struggling. For a vast majority of Spanish firms, human