Folia Microbiol. 36 (5), 468-477 (1991)

The Production of Extracellular and Intracellular Free Amino Acids during Aerated Fermentation of Glucose by Baker's Yeast (Saccharomyces cerevisiae) G.W. MALANEya~-, R.D. TANNERb*, and A.M. RODRIGUESc aDepartment of Civil and Environmental Engineeringy Vanderbilt University, Nashville, Tennessee 37235, USA bDepartment of Chemical Engineering~ Vanderbilt University, Nashville, Tennessee 37235, USA CUniversity of Blumenau (FURB), Blumenau, Santa Cotarina, Brazil Received January 7, 1991

During a study of the effects of a high level of NaCI on the content of free intracellular amino acids in baker's yeast grown in aerated fermentation of glucose it was found (Malaney et al. 1988, 1989; Malaney and Tanner 1988) that 0.6 mol/L exogenous NaCI significantly increased the content of free intracellular citruUine, glutamine, ornithine, arginine and lysine (all basic amino acids) over that observed at zero mol/L exogenous NaCI. (Exogenous is defined as salt added beyond that present in the mineral salts in the culture medium.) This paper describes the production and relative relationships of both extracellular and free intracellular amino acids by S. cerevisiae under conditions of high NaCI content in the growth medium at pH 5 and 32 ~ For early culture times (6 h), the production of glutamine, citruUine, valine, isoleucine, ornithine, lysine and histidine were all enhanced by the addition of NaCI. For late times (24 h), except for ornithine, the early-time-enhanced amino acids continued to be enhanced by the addition of NaCl. In addition, the yields of several other amino acids also were increased by exogenous salt at this late time. These include aspartic acid, threonine, glutamic acid, cystine, methionine, tyrosine, phenylalanine and arginine. ABSTRACT.

This paper is the fourth in a series (Malaney et al. 1988, 1989; Malaney and Tanner 1988) which reports on the effect of NaC1 on the production of amino acids in aerated S. cerevisiae. The first three papers deal with the content of amino acids in the intracellular pool in baker's yeast. Here, the effect of salt on the extracellular amino acids is featured and is compared with the corresponding intracellular amino acids (from the same runs), particularly to see if a type of "equifibrium" exists between the two locations. Physiological stresses, such as a high salt environment, cause free amino acids to leak out of yeast cells. Three areas of this phenomenon are important to study, (1) cell growth, (2) amino acid production, (3) cell product retention. Salt stresses the yeast cell membranes by altering their osmotic pressure. Under certain conditions (low aeration rate, ocean salt concentration, 32 ~ and pH 5), yeast ceils will yield a two- to four-fold increase in intracellular lysine levels between 8 and 12 h of culture time (Malaney et al. 1988). We are investigating how such salt alterations effect the "spillage" of these overproduced amino acids as well as other intracellular amino acids. Since these partitioned amino acids are separated by the yeast cell membrane, it is presumed that active transport mechanisms control the relative proportion of the intra- and extracellular amino acids. The effect of salt on that active transport mechanism is also presumed to account for differences in partitioning by the cell membrane. As there become fewer and fewer truly fresh-water sources in the world, brackish or salt waters will have to be used for irrigation, washing, drinking, and even fermentation processes. In this paper we explore whether such salty (in NaCI) water causes baker's yeast to significantly leak out its valuable content of amino acids, knowing from previous studies that NaCI can also enhance the intracellular content of lysine (Baker et aL 1985). If there is indeed significant leakage, the entire fermenta-

!

YDeceased, August 12, 1988. *To whom communications should be addressed.

1991

PRODUCHON OF FREE AMINO ACIDS 469

tion broth (yeast cells and extracellular liquid) could perhaps be added directly to animal feed, provided two constraints are met, viz. (1) the broth is blended with enough fresh water to keep the salt content below the maximum threshold (the Nutritional Constraint), and (2) no (expensive) trucking operation between the fermentor and the feed lot is required. In other words, the feed lot must be on the same grounds as the yeast plant as it is today in certain sugar plantations in Brazil (the Engineering Constraint) to prevent "spoilage" to the broth during transport. One possible bonus here is that a significant pollution problem of "dumping" the broth is turned into a useful and positive outlet.

MATERIALS

AND

METHODS

Descriptions of the fermentation microorganism used (Saccaromyces cerevisiae) and fermentation conditions are given in Park et al. (1985) and in Baker et al. (1985). The growth medium C of Maxon and Johnson (1953) was employed. The growth medium also contained 100 g glucose per liter of synthetic medium in a 1-L beaker. The aeration rate in all cases was 1.4 volume of air per volume of broth per minute (VVM). Sampling protocol. As soon as the yeast cell inoculum (ca. 2.5 g/L, dry mass) was well mixed with the growth medium, i.e., when clumps of yeast cells were no longer seen floating on the surface of the growth medium (15-20 min), a 2-mL sample of the inoculated growth medium was collected by means of a measuring pipette with a large orifice. Treatment of sample for analysis of intracellular amino acids. The 2-mL mash sample was pipetted into a test tube and capped with foil. The liquid level was marked on the side of the test tube. The sample was centrifuged at 1 075-1 200 g for 10 min. The supernatant fiquid was poured off and retained for extracellular amino acid analyses. If the supernatant was cloudy, centrifugation was repeated. The cell pellet was washed with ca. 2 mL water and centrifuged again. This supernatant was discarded. Then a 0.15 mol/L NaC1 solution was added up tG the mark on the test tube and the mixture was well shaken to resuspend the cells. This cell suspension was stored in the freezer at - 5 ~ for more than 24 h. At analysis time, the cell suspension was thawed and analyzed for amino acid content of the cells. Preliminary testing by us and others (Lewis and Phaff 1963; Heathcote et al. 1972) had shown that this freeze-thaw treatment released the free intracellular amino acids from S. cerevisiae as efficiently as did the conventional boiling of yeast cells. At the time the amino acid sample was collected, a 1-mL sample was removed for use in the determination of yeast cell mass concentration (Baker et aL 1985). Sampling was repeated, in general, every hour for the first 10 h and then at 12 and 24 h. Analysis of amino acid concentrations. Solid 5-sulfosalicylic acid was mixed with the frozen and thawed yeast cell preparation (50 g/L) to precipitate proteins. The sample was centrifuged and the protein-free supernatant liquid was analyzed for amino acid content in a Dionex semi-automated ion exchange chromatograph. Lithium citrate buffers were used. Four eluates at different pH and at different temperature were used during a run. A continuous concentration gradient did not form. The column was a Dionex D-300 physiological cation exchanger in a narrow bore steel casing. Fluoropa (OPA) detection was used with a Gilson Spectra/Glo fluorometer. Fluoropa reacts with any primary amine group to form a fluorescent complex. Reference chemical compounds were used to locate and quantify the chromatographic peaks. This method yields peaks for amino acids, some substituted amino acids, several amino acid precursors and several compounds related in structure to amino acids. In this paper, these compounds are referred to collectively as amino acids or as FIAA (free intracellular amino acids).

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G.W, MALANEY et aL

Vol. 36

RESULTS A N D DISCUSSION

The production of intracellular, extracellular and total free amino acids by S. cerevisiae grown in aerated Maxon-Johnson medium using 10 % glucose as substrate, in the presence of no and 0.6 mol/L exogenous NaCI is shown in Table I. In general, the production of intraceUular amino acids reached a peak at 8-10 h and then fell off, while the extracellular amino acids continued to rise for 24 h. The isoelectric points for the 22 common amino acids are also given in the table. Ammonia, (while not an amino acid) is also listed since it was a significant measured variable and it serves to help calibrate the system and develop material balances. A consideration of the total amino acid production (Table I) reveals that at 6 h, the production of basic compounds (glutamine, citrulline, ammonia, ornithine, lysine and histidine) was greater at 0.6 mol/L NaCI than without NaCI. The situation was reversed for the other amino acids. At 24 h, again the production of the basic compounds, except histidine and ornithine, was greater at 0.6 mol/L NaC1 than without NaCI~ Analysis of the intracellular amino acid data at 6 h shows that the production of only three amino acids - glutamine, citrulline and histidine - was greater for 0.6 mol/L NaCI than without NaCI, while after 24 h only the production of citrulline, ammonia and arglnine was greater for 0.6 mol/L NaCI than without NaCI. Consideration of extraceUular amino acid results reveals that at 6 h, the production of glutamine, citrulline, valine, isoleucine, ammonia, ornithine, lysine and histidine was greater for the higher NaCI level. At 24 h this list increased by the addition of aspartic acid, threonine, glutamic acid, cystine, methionine, tyrosine, phenylalanine and arginine, while ornithine dropped from the list. 1

I

1

-10gK

I

I

0 O

2

0 ~ 06A

~ 6

A

A A

I

I

I

:

%0

o~ 0 o

Lx 0

0 6

A

A

6 I

L

6

I

I

I

6

8

10

12 pl

Fig. 1. Effect of salt on the partition coefficient K (CFEAA/CFIAA) after 6 h (top) or 24 h (bottom) of fermentation as a function of pI; triangles, no NaCI added; circles, 0,6 mol/L NaCI.

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P R O D U C T I O N OF FREE AMINO ACIDS

471

In general, more basic amino acids were produced at 0.6 mol/L than without NaCI, regardless of classification: intracellular, extracellular or total amino acids. The other amino acids were produced more extensively without NaCI than at 0.6 mol/L NaCI. Comparison of the amounts of intracellular, extracellular and total free amino acids produced without and with 0.6 mol/L NaCI are shown in Tables II-IV. The amino acid production amount of concentration term is expressed by the change term, AC, which is the concentration of either the 6 h or 24 h point less the concentration of the 0 h point. The top seven amino acids listed in the no NaCI columns are those which showed the highest productions at the incubation periods indicated. The amounts of the reported free extraceUular amino acids (FEAA), relative to the free intracellular amino acids (FIAA), are expressed in Fig. 1, for 6 and 24 h of fermentation, by the ratio of their concentrations. The partition coefficient ratio K is calculated from: (1)

K : CFEAA/CFIAA,

where CFEAA data are taken directly from Table I. The C~TAAdata in Table I cannot be used directly since they represent the "spilled" intracellular free amino acid concentrations in the extracellular broth sample volume. Of the 19 common amino acids, the K of histidine was not graphed because the extracellular levels were so low. The K's for phenylalanine and tyrosine were plotted when the extracellular

100

I

10--

1

I

I

A .L -0

0

0.1 100

Lys

I

o

o Arg

x30rn

O0

I

I

t

t

10 o Lys Asp

1

~176 o o

o 0rn

A[o 0.1 2

I t,

I 6

I 8

o Arg

I 10

pl

12

Fig. 2. The ratio Q (Ks+/Ks_) after 6 h (top) or 24 h (bottom) of fermentation as a function of pl; triangles, no NaCI added (KS_ ); circles, 0.6 mol/L NaCI (Ks +).

concentration levels were sufficiently high. Instead, C'FIAA data are taken from Table I of Malaney et al. (1988), where the Table I C'FIAA data have already been divided by the dry cell concentration at that particular time and are in units of mg amino acid per g of dry yeast cells. These C'FIAA values are

472

G.W. MALANEY

et al.

Vol. 36

T a b l e I, C o n c e n t r a t i o n s ( m g / L ) o f i n t r a - a n d e x t r a c e l l u l a r a m i n o acids a in 1 L c u l t u r e , w i t h o u t a n d w i t h e x o g e n o u s 0.6 m o l / L N a C I in M a x o n - J o h n s o n c u l t u r e m e d i u m

Exogenous amino acid

Incubation time, h NaCI 0

2

4

6

8

10

24

I E

12.2 2.1

14.2 1.4

14.4 7.5

26.1 3.7

80.0 2.0

T

14.3

15.6

21.9

30.4

82.0

I

0.9

5.1

7.2

19.7

-

51,2

26.7

E T

1.3 2.2

1.9 7.0

3.6 10.8

4.7 24.4

-

3.9 55.1

7,7 34.4

A s p a r t i c a c i d ( p I = 2.77) without

with

-

68.1 4.5 72.6

2-Aminoadipic acid (pI = 3.18) without

with

I

~0.4

2.7

6.2

7.9

1.6

-

5.3

E T