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Enhancement of Yeast Fermentation by Addition of Oxygen Vectors in Air-Lift Bioreactor
SHIRU JIA,’ MINGXIA WANG,’ PRIHARDI KAHAR,Z YONGSOO PARK,2 AND MITSUYASU OKABE** Department of Food Engineering, Tianjin Institute of Light Industry, 1038 Dagu Nanlu, Tianjin, China’ and Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422, Japan*
Received 13 January 1997/Accepted 2 May 1997
The effect of an oxygen vector on the oxygen transfer rate in air-lift bioreactor was evaluated using the ratio of the volumetric oxygen transfer coefficient to the volumetric fraction of the oxygen vector. When n-dodecane and perfluorocarbon were added at final concentrations of 3% and 2% (v/v), respectively, the oxygen transfer rate reached a maximum value. By addition of 3% (v/v) of n-dodecane to the yeast fermentation, the yeast concentration increased to 26.2 g/Z which was 20% higher than in the case of fermentation in the absence of the oxygen vector. [Key words:
oxygen vector, n-dodecane, perfluorocarbon, air-lift bioreactor, yeast]
Since oxygen vectors, generally organic compounds, have a higher oxygen solubility than water, organic compounds such as perfluorocarbon (l), n-dodecane (2), nhexadecane (3), liquid hydrocarbons (4), and soybean oil (5), can be used as oxygen vectors. We previously reported that alkanes from Cl2 to C16, byproducts of distillation processes in the petroleum industry, could be used as oxygen vectors (4). Addition of an alkane mixture to a final concentration of 4% increased the volumetric oxygen transfer coefficient and yeast growth rate by 1.26and 1.3-fold, respectively, compared with in the case of fermentation in the absence of an oxygen vector (4). The advantage of using oxygen vectors in fermentation that they increase in the oxygen transfer rate from the gas phase to the microorganisms without the need for extra energy supply. Oxygen vectors can act as surface active agents to lower the surface tension of water and increase the gaseous specific interfacial area. To date, most work concerning the enhancement of oxygen transfer from the gas phase to the microorganisms using oxygen vectors has been conducted using stirred reactors. In the case of air-lift bioreactors or bubble columns, the oxygen transfer rate is lower than that in the case of stirred reactors. Therefore, the use of oxygen vectors in a bioreactor which is operated under low agitation conditions enhances oxygen transfer. However, sometimes the enhancement of oxygen transfer by oxygen vectors in an extraction loop bioreactor is limited by the coalescence rate of the oxygen vectors droplets at the bottom of the reactor (6). Although there have been many studies using air-lift bioreactors, there are few reports regarding oxygen transfer in air-lift bioreactors containing oxygen vectors. In this study we investigated the effect of oxygen vectors in an air-lift bioreactor on enhancing yeast fermentation and aimed at increasing the yeast production rate. n-Dodecane was purchased from the Chemical Co. Shanghai (China) and its physical properties at 30°C were as follows: density, 743 kg/m3; surface tension, 24.6dyn/cm; interfacial tension with respect to water, 32.9 dyn/cm; boiling point, 214’C; oxygen solubility (35”C, atmospheric pressure), 54.9 mg/l. * Corresponding author.
Perfluorocarbon was obtained from the Chemical Co. Shanghai (China) and its physical properties at 30°C were as follows: density, 1,750 kg/m3; surface tension, 17.8 dyn/cm; interfacial tension with respect to water, 25 dyn/cm; boiling point, 206”C, oxygen solubility (35”C, atmospheric pressure) 118 mg/l. Saccharomyces cerevisiae AY-12 (Tianjin Institute of Light Industry, Tianjin, China) was used in this study. This strain cannot use oxygen vectors (n-dodecane or perfluorocarbon) as sole carbon sources. A loop of S. cerevisiae was inoculated into 100 ml of the preculture medium in a 500-ml Erlenmeyer flask, and grown in standing culture at 30°C for 12 h. An aliquot of the culture (5OOml) was then inoculated into 4.5 I of the production medium in an air-lift bioreactor for the production of yeast. The culture temperature was controlled at 34”C, and the pH was maintained at 5.0-t0.5. The air-lift bioreactor consists of three parts, a conical bottom holding the sparger, a cylindrical part containing a draft tube, and a top portion with a degassing zone. The total length of the reactor is 610mm. The length and diameter of the cylindrical part are 500 and 120 mm, respectively; those of the draft tube, 350 and SOmm, respectively. In order to avoid nutrient depletion during the culture, the sugar concentration was measured every 30min, and then a concentrated sugar solution containing 40% sucrose and 2% yeast extract was intermittently fed into the bioreactor in proportion to the sugar consumption rate. During the culture the reducing sugar concentration was maintained at about 5 g/l. The dissolved oxygen concentration in the air-lift bioreactor was measured with a DO meter (Tianjin Institute of Light Industry). The DO sensor was located at 250mm from the top of the reactor. The DO concentration measured in the reactor was 10 s delay at the condition of viscosity of below 1Ocp for mixing uniformly (7). The aeration rate in the air-lift bioreactor varied from 0.5 to 2.0vvm. When the aeration rate was high, the working volume decreased due to evaporation. To maintain the culture volume at 5 I, a level sensor was used to feed sterilized water into the reactor. The preculture medium contained 12°C Brix wort. One kilogram of concentrated malt extract was diluted with 2.6 I tap water to yield 12“ Brix wort. The diluted malt
The culture broth was filtered through filter paper (Xinhua no. K2HP04. Eq. The effect of oxygen vector concentration on the ratio of volumetric oxygen transfer coefficient is represented as follow. HjP04. Reducing sugar concentration was measured using the Somogyi-Nelson method (8). 0. Denmark) and maintaining the mixture at 60-7O’C for 30min. = pc*o i. According to the volume-average rule of the dispersion. In view of the result of Rols et al. and then adding 1 ml of a-amylase (Termamyl.) filtered.. n-Dodecane (A) and perfluorocarbon(B) wereused as oxygen vectors.. Rols’s experiments (2) on the rheological behavior of these emulsions indicate that in the (1) FIG. (kLa) / (kLa)pp=oincreases with the increase in ‘p. and then at 60°C for 3 h. and when S. 2. Eozmm reached a constant value. and passed through filter paper (Whatman no. value is 8. when (0>2% and 3%. (NHJ2S04. n-dodecane. 84.=gwo-((Ioo+fow). 1 1 (2) Since the saturated oxygen concentration in aqueous solution is much higher than that in an aqueous solution. The corn syrup was prepared by mixing one kilogram of corn powder with 3 1 of tap water. The oxygen transfer rate in air-lift bioreactors is relatively low compared to that in jar-fermentors.C*. 3 can be modified to c*. 2 can be modified to. 2 gives. it began to coagulate at the bottom of air-lift bioreactor. (2). The spreading coefficient was measured by the method of Rols and Goma (5). The volumetric oxygen transfer coefficient kLa was measured by the method of Dang et al.=O in Eq. when ‘F>2% for n-dodecane or 3% for perfluorocarbon. the diluted malt extract was autoclaved at 120°C for 15 min.. Eo2 is defined an indicator of oxygen transfer enhancement and is represented as Eo2= (kLa). Effect of volume fraction of oxygen vectors on the volumetric oxygen transfer coefficient and its ratio. In the case of n-dodecane and perfluorocarbon as oxygen vectors at ‘p (volumetric fraction of the oxygen vectors)<2% (pertluorocarbon) and 3% (n-dodecane). the a-amylase is deactivated in 0. [ (kLa). 2. When S. For example.&C*1 (b4.=9 in Eq.7 x lop3 for ndodecane and 1. Surface tension was measured by the ring method using a surface tension meter (4). 3 Hangzhou. Effect of volume fraction of oxygen vectors on the maximum enhancement of oxygen transfer in the air-lift bioreactor. Aeration rate was tied at 2 vvm. E 02max= (kLa). when the oxygen . Thereafter.VOL. 8. The relationship between EQ. When the (o value was less than 2%. Although n-dodecane has a positive spreading coefficient. The wet mycelia on the filter paper were washed twice with saline water and then four times with distilled water before being dried at 100°C for 4 h.=&*.. perfluorocarbon was mixed completely. (kLa). there is a notable increase in the apparent viscosity.8 3 120 NOTES 177 80 PC%) 9. During the experiment we observed that the bubble size distribution was affected by the addition of the oxygen vectors in the dispersion. The results were identical to those reported previously (4).=o= c*w p3)c*w Substituting C*. Symbols: o . broken slope region. 1997 1. which might affect the oxygen transfer rates. [(E. 3) to remove the resulting precipitate. After this incubation. Cell concentration was calculated as dry cell weight. NOVO NORDISK Co. C*.45 x lop2 for perfluorocarbon. China). 200ml (with 25% reducing sugar). This may cause a decrease in kLa. periluorocarbon. and C*.8 ml (50%). Perfluorocarbon is difficult to disperse in the bioreactor due to its high density. 0 4 8 y?CW 12 16 extract was incubated at 45°C for 2 h. EQ mm increased. 25 g.1 MPa.(C* . but at ‘p higher than 3%.. urea. 1. and C*. oxygen vectors will simply form floating lenslike drops. The production medium consisted of the following ingredients (g/E): corn syrup. Q+.:J and is shown in Fig. it has the same properties in air-lift bioreactor as liquid hydrocarbon with a negative spreading coefficient (4).7 g.(1 c*. After saccharification at 55-60°C with addition of NovozymTMAG (NOVO NORDISK Co. (9). The S.4g.>O the oxygen vector will spread on a water surface.o reached a maximum value and then decreased. and cpis shown for various of oxygen vector volume fractions in Fig.<O.=o (3) where Eo2 mm denotes the maximum enhancement of oxygen transfer.0. the spreading coefficient of an oxygen vector S. 1. 3. 2. In order to compare the effects of different oxygen vector volume fractions on the rate of oxygen transfer from a gas to a liquid phase. As (o increased. 4 into Eq. (%I FIG./(kLa).
Bioeng. .. Park. Biotechnol. C. M. Dang... 26. A. Chem.. and Coma. B. Park. % interfacial tension between i and j components. J. J. 78. It was concluded that in the yeast fermentation. 35.2. Although the Eoz increased 2-fold by compared to that obtained in the absence of the oxygen vectors. G. the mean bubble diameter (measured by a photographic method) was reduced by 15% from the control. Li. 82.. respectively. If any culture that is sensitive DO would be tested. Y. Karrer. h-l EOZ : enhacement of oxygen transfer.. J. Rols.: Twoliquid-phase bioreactors.: Oxygen transfer enhancement in aqueous/perfluorocarbon fermentation systems. Biotechnol. D. T. The ndodecane was added to the fermentation broth at 0 h and 6 h. % c* : saturation dissolved oxygen concentration. 3. H. The effect of 3% n-dodecane on the yeast fermentation in the air-lift bioreactor is shown in Fig. L.20 wm. D. Condoret. 0.0 and 21. 3) the yeast concentration reached maximums of 26.8 g/l corresponding to 2. and Okabe. s. addition time is 6 h. A. 265268 (1994). Ferment.20 vvm. S. 3... Junker. N.: Enhancement of glucose oxidase fermentation bv addition of hvdrocarbons.: Mechanism of enhanced oxygen transfer in fermentation using emulsified oxygen-vectors.. FERMENT.: Enhanced oxygen transfer in tower bioreactor on addition of liquid hydrocarbons. J. 578-585 (1990). After 13 h of cultivation (Fig. 427-435 (1990). Y... Bioeng.. This indicates that this fermentation was not critical to DO. but the gaseous interfacial area increased in proportion. 6. 61 (1945). B. C.68 g/l/h average yeast production rates. H. N.. This experiment yielded two facts. and Chen. S.00 and 1. Concentrations of yeast (A) and DO (B) in the presence and absence of n-dodecane in the air-lift bioreactor. Enzyme Microb.. 3. 35. A.. addition of n-dodecane to the fermentation broth resulted in a 20% increase in yeast concentration. H. P. Rols.. Biol. H. M.: Neomycin production by partial immobilization of Streptomyces frudiae on cellulose beads in an air-lift bioreactor. Biotechnol. S.. max: maximum Uij * e REFERENCES 0 2 4 6 8 10 12 14 1. 298-303 -(1994). 853-865 (1977). and Dynn. 2. 7-12 (1991). T. 2. L. Experimental observations. Ferment. Bioeng.: Oxygen transfer coefficient by dynamic model moment analysis. and Tramper. Second. I. NOMENCLATURE DO : dissolved oxygen concentration.. the time of addition ndodecane to the broth had no effect on the yeast growth. S.. 160. Ferment. J. the effect of the oxygen vector will be shown much clearly. addition of 3% n-dodecane to the fermentation broth had a positive effect on yeast growth. First.178 JIA ET AL. Lett. T. Sonsbeek.02. G. : spreading coefficient.. 15. 191-193 (19%). J. N/m (Subscript) G: gas. H. BIOENG. N/m IO : volumetric fraction of the oxygen vector. 5. Beeftink. 722729 (1993). J. and Okabe. The positive effect of n-dodecane on the yeast yield was attributed to an elevation in the dissolved oxygen concentration. W: water. Biotechnol.. J. Bioeng. Hatton. g/m3 * actual dissolved oxygen concentration. I. 60. Bioeng. and Goma. 0: oil. J. no addition. I. Time (h) FIG. L. addition time is 0 h.. Bioeng. M. vector volume fraction was 5% (v/v). All the runs were carried out at an aeration rate of 1. D.: Enhanced oxygen transfer rates in fermentation using soybean oil-in water dispersions. 13. Technol. Jia. Fonade. 4. This is probably because the DO in the culture broth was not depleted during the culture with or without the oxygen vector (the lowest DO was 10%). g/m3 c kra i volumetric oxygen transfer coefficient. Ohta. . Aeration rate was 1. 78.. Li. Somogyi-Nelson: A new reagent for the determination of sugars. R. and Wang. the yeast concentration was only improved by 20%. J. Symbols: 0.