Biomass Yields and Energetic Yields of Oleaginous Yeasts in Batch Culture

Jae G. Pan and Joon S. Rhee* Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, P. 0.6. 150, Chongyang, Seoul, Korea

Accepted for publication March 2 I , 1985

This article provides the analysis on biomass yields and energetic yields of the oleaginous yeasts. The biomass yields of the oleaginous yeasts are consistently lower than nonoleaginous microorganisms, whereas their en- ergetic yields are higher. Data inconsistencies of literature are explained by the variation of energy contents of olea- ginous yeasts.

The oleaginous yeasts have the capability of ac- cumulating up to 70% intracellular lipid under nitrogen- limiting and other lipid-accumulating conditions. ’,’ Quantitative physiology of these yeasts was based on a rather unsystematic approach. Lipid yield, g lipid/100 g substrate, has been used as a means of expressing the oleagenicity.’ However, the close analysis of the literature on the oleaginous yeasts revealed that biomass yields were abnormally low, especially under the con- dition of lipid accumulation. In order to analyze the problem posed more critically, mass and energy balance method^^.^ should be used because energy contents of oleaginous yeasts increase with the increasing content of intracellular lipid.’ Generally, the energy contents of microbial cells could be assumed to be however, not in the case of oleaginous yeasts. In our laboratory a chemostatic approach to Rhodotorula glutinis was carried out to determine the physiological parameters, which resulted in rather low yield, 0.244 g biomass/g glucose.*

Thus, the purpose of this article is to analyze the biomass yields and energetic yields of oleaginous yeasts based on energy contents of these cells. The data analyzed will show that the biomass yields of oleaginous yeasts are much lower than nonoleaginous microor- ganisms, whereas energetic yields are higher.

Method of Data Analysis

CH,O,N, is written The reductance degree (y) of the chemical compound

(1) y = 4 + p - 2n - 3 q

when the nitrogen source is NH3.3 * To whom all correspondence should be addressed.

Biotechnology and Bioengineering, Vol. XVIII, Pp. 112-1 14 (1986) 0 1986 John Wiley & Sons, Inc.

Based on the balance equation for microbial growth energetic yield 7 is defined3s4

71 = (z) y x / s

where YXls is the biomass yield in g biomass/g substrate; c b and us are the weight fractions of carbon in biomass and substrate, respectively; and Y b and ys are the re- ductance degreee of biomass and substrate, respec- tively. The energetic yield is the ratio of the heat of oxidation of the biomass produced to the heat of ox- idation of the substrate utilized where the oxidation results in the production of CO, , H20, and NH3.4 Eroshin and Krylova measured fib and Y b of oleaginous yeask5 Regression analysis of their data shows the following linear relationship between lipid content and

(3) where L is lipid content (% g lipid/g biomass).

Lipid yield, g lipid produced per 100 g substrate, has been used as an index of lipid formation efficiency.

(4)

WbYb

(TbYb = 1.6 + 0.0308L

Lipid yield = L . Yx,,

Replacing equation ( 2 ) and ( 3 ) into ( 4 ) gives

For the analysis of the batch data, we excluded the data from static cultures, the data that showed the inconsistencies in their own presentation, the data from unusual lipid-accumulating conditions, and the data that did not specify NH3 as the nitrogen source. All of the carbon sources are carbohydrates that have mSy, of 1.6-1.68. Starting with lipid content, c b y h was cal- culated from equation (3), which was then used for the calculation of 71 by equation ( 2 ) .

RESULTS AND DISCUSSION

Since equation (3) was obtained by Eroshin et al.’ from the data based on the ethanol-grown oleaginous

CCC 0006-3592/86/010112-03$04.00

yeasts, the validity of the equation was checked the- oretically for glucose-grown oleaginous yeasts. If all of the biomass was the lipid (100% lipid content), then obyb equals 4.68 from equation (3) and leads to lipid yield of 34.1 for the case of glucose (uSyS = 1.6). For the total conversion of glucose to trioleoylglycerol, Ratledge calculated the maximum lipid yield based on biochemical pathway to be 32.9,* which is fairly close to the 34.1 obtained from equation (5) . In view of heterogeneity of yeast lipids (although the major fraction is triacylglycerol), it was reasonable to use equation (3) for the further analysis. It is rather surprising to see that the macroscopic method gives similar value of lipid yield, because it exemplifies the usefulness of macroscopic method in the approximation of yield.

Biomass yield, Yxls, was plotted against lipid content in Figure 1. The maximum yield [when r ) = 1 in eq. (2)] decreases with the increase of lipid content. The data collected clearly show that the biomass yield decreases with the increasing content of lipid. The cells of 50% lipid content and Yxls of 0.356 have the same r ) (= 0.7) with the cells of 10% lipid content and Yx,s of 0.59. As is evident in Figure 1, it would be wrong to state that "high values of lipid content has been possible only with very poor yield,"' because this statement implies that the efficiency of microbial growth is adversely affected by the lipid accumulation. As is shown by theoretical lines in Figure 1, the average of energetic yield (= 0.678 & 0.109) is quite high as compared with that of nonoleaginous microorganisms (= 0.55).6 However, average biomass yield (= 0.343

.9 , 1

.7 h

0 0

.1 I I I I I I I I 10 20 30 40 50 60 70 80

Lipid content. %

Figure 1. Biomass yield as a function of lipid content. Data from 21 sources on the lipid yield and biomass yield available since 1949 were included.

I

0 1 1 I I I 1 I

10 30 50 70

Lipid content. '%

Figure 2. Change of lipid yield as a function of lipid content.

k 0.086) is much lower than that of nonoleaginous microbial cells (= 0.5 - 0.55). At present, it is not clear whether r ) is maintained at a constant level with the increase of lipid content or is dependent on lipid content.

In Figure 2, the lipid yields were correlated with the lipid contents. As is shown by the theoretical lines, the increasing trend of lipid yield is evident with the increase of lipid content. It seems that r) does not change significantly with the increase of lipid content, though the experimental data are rather scattered.

With the above data analysis in mind, it should be interesting to reassess the previously published yield di~crepancies'.~ and the process yield which was used for the single-cell oil (SCO) process economics.'

In a recent article Yamauchi et al.9 cultivated Li- pomyces starkeyi in fed-batch culture system and ob- tained a very high concentration of lipid, 83 g/L, at 140 h culture. However, they noticed the decrease of biomass yield with the increasing culture time, which was explained to be possibly due to the accumulation of high-energy substances. It was quite normal because the energy content of L. starkeyi increased with the accumulation of lipid. Interestingly, the increase of r )

is observed in the lipid-accumulating stage when re- calculating their data (Fig. 10 of ref. 9).

Ratledge considered the possible biomass yield and lipid content of SCO process to be 0.55 and 40%, respectively.' Similarly in our work' a batch result yielded the biomass yield of 0.47 and the lipid content of 54%. These two sets of data lead to the r) of 0.97 and 0.96, respectively, which are too high to be rea- sonable. Quantitative physiological approach with Rhodotorulu glutinis" resulted in rather low biomass yield (0.244) in continuous culture, which was lower than that in batch culture.' Obviously, it was an error because maximum Yxls in continuous culture must be higher than that in batch culture. Such an error was inevitable where the energy content of cells was as-

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sumed to be constant, the assumption of which, how- ever, is valid for nonoleaginous microorganisms.

It is interesting to note that 7 under nitrogen-limiting condition in continuous culture showed consistently higher average value (= 0.718 & 0.049) than 7 under carbon-limiting condition (= 0.609 k 0.026) (calculated from ref. 11) However, this trend should be confirmed both in batch and continuous culture because their data were significantly inconsistent (e.g., 7 > 1 for D = 0.06/h). For this reason, a series of carefully designed experiments are required to understand whether 7 is consistently high for the oleaginous yeasts or for the lipogenic conditions. These experiments are underway in our laboratory. High value of 7 is correlated with the high value of Yxl0 (g biomass/O, consumed).394 It was measured in continuous culture'0.'2 for Candida 107 and R . glutinis. The specific oxygen uptake rate under lipid-accumulating conditions was much lower than under nonlipid-accumulating conditions, which could be a partial evidence for 7 dependence on li- pogenic conditions, not on particular strains. This fact may be an advantage of SCO process over the other

bioprocesses since the bioreactor productivity is usually limited by oxygen transfer rate.

References 1. M. Woodbine, Prog. Znd. Microbiol., 1, 179 (1959). 2. C. Ratledge, Prog. Znd. Microbiol., 16, 119 (1982). 3. I. G . Minkevich and V. K. Eroshin, Folia Microbiol., 18, 376

4. L. E. Erickson, I. G . Minkevich, and V. K. Eroshin, Biotechnol.

5. V. K. Eroshin and N. I. Krylova, Biotechnol. Bioeng., 25, 1693

6. J . A. Roels, Biorechnol. Bioeng., 22, 2457 (1980). 7. A. A. Esener, J. A. Roels, and N. W. F. Kossen, Biotechnol.

8. S. H. Yoon, J. W. Rhim, S. Y. Choi, D. D. Y. Ryu, and J .

9. M. Yamauchi, M. Mori, T. Kobayashi, and S. Shimizu, J .

10. S. H. Yoon and J. S. Rhee, Process Biochem., 18, (Oct.), 2

I t . C. 0. Gill, M. J. Hall, and C. Ratledge, Appl . Env. Microbiol.,

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114 BIOTECHNOLOGY A N D BIOENGINEERING, VOL. 28, J A N U A R Y 1986