Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S413

Figure 1. 1,3-Propanediol-production of AKR91b, AKR92a and AKR102a with 60 g/Lpure glycerol ( ) and 30 to 80 g/L initial 1,3-propanediol ( ).

potential producers as Clostridium butyricum (GRAS) facilitating anapplication on industrial scale.

The small-scale experiments allowed for a pre-selection reduc-ing the number of strains to be tested on fermentation scaledrastically where AKR102a demonstrated the best production char-acteristics. A process optimization is currently conducted.

An adaption of the enrichment conditions and evaluationparameters to desired strain traits such as usage of a specific crudeglycerol or nutrient requirements is possible as well as a trans-fer of this screening method to different substrates or additionalanaerobic end-products as biobased chemicals.

doi:10.1016/j.jbiotec.2010.09.557

[P-I.192]

Identification of Cost Drivers and Subsequent Economic Optimi-sation of 1,3-Propanediol Production

E. Mildner ∗, A.K. Ringel, T. Willke, K.-D. Vorlop

Johann Heinrich von Thünen-Institut (vTI), Institute of AgriculturalTechnology and Biosystems Engineering, GermanyKeywords: 1,3-propanediol; crude gycerol; fermentation; yeastextract substitute

The microbial production of 1,3-propanediol (PDO) from glyc-erol is currently not economically competitive with conventionalchemical production. Raw materials for the fermentation mediumand glycerol are the main cost drivers and account for more than50% of the total costs. Hence, cost reduction can be achieved eitherby using crude glycerol from biodiesel production or replacing cost-intensive media components such as yeast extract.

Screening tests in-house provided multiple gram-positive,spore-forming bacterial strains capable of producing PDO fromcrude glycerol with high product tolerance, productivity and yield.

Under optimized conditions using pure glycerol and yeastextract one strain was able to produce 95.6 g/L PDO within 43 hours.When pure glycerol was replaced with crude glycerol 78.5 g/L PDOcould be obtained within 48 hours (Fig. 1a). Additionally a scale upfrom 0.7 to 10 L exhibited no problems.

With yeast extract being a main cost driver the need to replacethe former arises. Residues from agriculture and animal feed indus-try represent suitable and inexpensive complex substrates for thistask. Preliminary experiments were carried out in microtiter scale.Promising substrates were additionally tested in batch fermenta-tions on pure glycerol. Using soy-based material it was possible toreach up to 70 g/L PDO without any yeast extract, however at aprolonged lag-phase. By adding small amounts (0.5 g/L) of yeast

Fig. 1. (a) PDO-production (fed-batch) using yeast extract and pure (square) orcrude glycerol (circle). (b) PDO-production (batch) using pure glycerol and soy-based material (star) or soy-based material plus yeast extract (triangle).

extract the lag-phase could be reduced from approximately 10 to4 hours also resulting in an increase of productivity by at least 100%(Fig. 1b).

The combination of all described approaches will result in arobust industrial process with significant cost reduction. Provideda crude glycerol price of 200 D per ton, an overall PDO price lessthan 1000 D per ton is achievable.

doi:10.1016/j.jbiotec.2010.09.558

[P-I.193]

Modelling of Glycerol to 1,3 Propanediol production

Ashok Kumar Srivastava ∗, Gurtej Singh, Guneet Kaur, AbhishekNigam, Subhash Chand

Indian Institut of Technology Delhi, IndiaKeywords: 1,3 Propanediol fermentation; Modelling; Fed-batchcultivation; Batch cultivation

1,3-propanediol production by fermentation has been a matterof increasing interest in recent years, because of its usage for thesynthesis of polyesters and biodegradable plastics. Its production isparticularly important as presently it can be produced from renew-able resources e.g. glycerol. Glycerol is mainly released from fatsaponification (the principal product being the free fatty acids usedfor detergent manufacture). With the production of 10 kg of esterfuel from rapeseed oil, 1 kg of glycerol becomes available. Thereforedue to increased production of plant oils in the developing coun-tries, glycerol is becoming available in surplus in the world market,which has led to a continuous reduction in its prices. In addition,the direct use of the crude glycerol from biodiesel transesterifica-tion process without any pre-treatment adds to the simplicity andprice relaxation of the process. Biotechnological processes need tobe developed to convert this substrate (glycerol) into higher valuedproducts such as 1,3-propanediol.

In the present investigation production of 1,3-propanediol wasattempted by bioconversion of Glycerol by Clostridium butyricum.Statistical media optimization was done for the growth and prod-uct formation by Clostridium butyricum. The batch cultivation of1,3-propanediol was then carried out in a 7 L (working volume4 L) (Applikon 1025) bioreactor with the statistically optimizedmedium composition and environmental conditions. The temper-ature, pH of the media and agitation rate were set to 37 ◦C, 7.55and 250 rpm respectively. Nitrogen gas was sparged at 0.1 vvm inthe bioreactor. The average product concentration, yield and pro-ductivity of the product were 46.1 g/L, 0.439 gm/gm of glycerolused and 1.65 g/L.hr respectively. This batch kinetic data was thenused for the development of a mathematical model. The param-eters of the model were identified by minimizing the differencebetween the model simulation and original experimental data. The

S414 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

model was then extrapolated to simulate different fed-batch oper-ating conditions. The potential use of the model to identify rightnutrient feeding strategies for fed-batch cultivation to improve theproductivity of 1,3-propanediol will be demonstrated.

doi:10.1016/j.jbiotec.2010.09.559

[P-I.194]

Development of a culture strategy to produce a bacteriocin typesubstance utilizing a strain of Enterococcus mundtii

Juan Carlos Gentina, Carlos Padilla, Paola Poirrier ∗

Pontificia Universidad Católica de Valparaíso, ChileKeywords: Bacteriocins; Enterococcus mundtii; Culture strategy

Lactic acid bacteria produce a variety of bacteriocins, peptidemolecules that act inhibiting growth of Gram-positive bacteria. Thestrain of Enterococcus mundtti used in this work was isolated at asalmon processing factory located southern Chile. Growing in MRSmedium it exhibits a high production capacity of a bacteriocin typesubstance (BTS) very active against Listeria monocytogenes.

Previous studies using MRS medium shown that BTS is producedgrowth associated obtaining a maximum volumetric productivityof 3.74*108 (AU/L*h) at pH 6.2 and 30 ◦C.

The objective of this work was to develop a fermentation strat-egy to maximize the volumetric productivity of BTS using MRSmedium.

Experiments were conducted batch wise in a 2 L bioreactor usingMRS medium, at 30 ◦C and 1 vvm. The agitator was a Rushton typeturbine operated at 300 rpm.

Initially four fermentations were run intending to evaluate theeffect of aeration and pH control policy on BTS productivity. Firstand second runs were aerated and pH controlled automaticallyadding KOH in one case and using phosphate buffer in the other.Third and fourth runs were non-aerated and the same pH policyas before respectively. The condition using aeration and phosphatebuffer gave the best results of BTS volumetric productivity equalto 1.87109 (AU/Lh) and a product yield of 6.8109 (UA/gcel). It wasobserved that high BTS production was stimulated by pH fall from6.2 to 4.8 during growth phase.

These results allowed designing a fermentation strategy con-sisting of a two stage batch operation. The first stage was aeratedand pH controlled automatically and the second also was aeratedbut without pH control. BTS volumetric productivity was 2.171010

(AU/Lh) and product yield was 5.21010 (UA/gcel).By using this strategy volumetric productivity was increased by

a factor of 11.6, using in both cases the same MRS culture medium.

Acknowledgments

PUCV Proyect BIOACTIVES, FONDEF PROYECT D04I1153.

doi:10.1016/j.jbiotec.2010.09.560

[P-I.195]

Condition of Mild Hypothermia does not Promote an Increasein Specific Productivity of Recombinant Protein in ContinuousCulture of CHO Cells

S Becerra 1,∗, M Vergara 1, R Gozález 2, N Osses 1, C Altamirano 1

1 Pontificia Universidad Católica de Valparaíso, Chile2 Rice University, United StatesKeywords: CHO Cells; Continuous Culture; Sub-physiological tem-perature; Proteomics

Mammalian cells for recombinant protein production areusually grown at 37 ◦C. However, mild hypothermia condition(28–34 ◦C) often leads the production improvement. Grow atlow temperatures can improve the maintenance of cell viabil-ity, decrease the consumption of carbon and energy sources andalso decrease the protease and sialidase activity. Nevertheless, themechanisms regulating these phenotypes are yet poorly under-stood.

Chinese Hamster Ovary (CHO) cells, producing human recom-binant tissue plasminogen activator (tPA) were grown in HyCloneCDM4CHO medium supplemented with 10 mM of glucose and6 mM of glutamate using spinner flask adapted to continuous cul-tures (140 ml) at 37 ◦C and 33 ◦C under controlled atmosphere bythe same dilution rate (D = 0,0167 ± 0,0007 h−1). Number of viablecells was determined using a haemocytometer and trypan blueexclusion method. After 3 time of residence we considered station-ary state and we take samples of cells and supernatant.

The main metabolites were measure: glucose, lactate andglutamate concentration using biochemical analyzer (YSI2700),ammonium concentration by the Berthelot reaction. Secretedand intracellular tPA concentrations were quantify via ELISA kit(Imulyse t-PA, Biopool) and identified through westernblot. ThemRNA-tPA was quantified by means of RT-PCR. The differentialprotein expression was investigated using 2D-DIGE and mass spec-trometry.

Our results demonstrate that the Ylac/glc was not affected bythe decrease of temperature, while the consumption of glutamateremarks decreased to 33 ◦C, but not the production of ammonium.Specific production of tPA in cells growing at mild hypothermiacondition but at the same specific growth rate is 67% lower thanspecific production at 37 ◦C; this is consistent with our results ofquantification of mRNA-tPA.

Then, we conclude that a decrease in temperature is not respon-sible for the increase in specific productivity of recombinantprotein, would rather a decrease in specific growth rate that occursin batch culture.

doi:10.1016/j.jbiotec.2010.09.561

[P-I.196]

Optimization of Exo-Polygalacturonase Production fromOrange Peel by Aspergillus sojae

AO Buyukkileci 1,2,∗, C Tari 1, HM Fernandez-Lahore 2, H GenckalDemir 1, N Gogus 1

1 Izmir Institute of Technology, Turkey2 Jacobs University Bremen, GermanyKeywords: Polygalacturonase; Aspergillus sojae; optimization; sub-merged fermentation

Pectinases catalyze the degradation of pectic substances, thusthey are used extensively in fruit juice and wine industry to facil-