421
Biotechnol. Prog. 1990, 6, 421-424
Effect of Acetaldehyde on Growth, Substrates, and Products by Leuconostoc mesenteroides ssp cremoris P. Schmitt and C. Divi&s* DBpartement de Microbiologie-Biotechnologie, ENS-BANA, Campus Universitaire Montmuzard, 21000 Dijon, France
Addition of 5.6 mmol/L of acetaldehyde to glucose broth led to an increase in the specific growth rate, a decrease in the specific glucose consumption rate, and an increase in the specific lactate production rate of Leuconostoc mesenteroides ssp cremoris. The amount of acetate produced was enhanced. For cells growing in broth containing glucose plus citrate, addition of acetaldehyde did not stimulate growth and did not significantly change the maxima of specific consumption rate of citrate and glucose; however, a change in kinetics was observed: the maxima appeared earlier in the presence of acetaldehyde than in its absence. In the absence of acetaldehyde, the specific citrate utilization rate was higher than the specific glucose consumption rate.
Introduction Leuconostocs are used in the dairy industry for enhancement of the aromatic and flavor properties of fermented milk products. Ethanol, acetaldehyde, acetate, diacetyl (2,3-butanedione), and acetoin (3-hydroxy-2-butanone) participate in aroma and flavor of fermented milk products. Acetaldehyde formed during the growth of lactic streptococci (1-3) is responsible for a green or yogurtlike flavor defect found in cultured dairy products (I ,4-6). In mixedstrain cultures, it has been suggested that Leuconostoc mesenteroides ssp cremoris utilizes the acetaldehydeproduced by the lactic streptococci (1,6). The effect of addition of acetaldehyde to various strains of starter bacteria has been studied by several workers, but the effect on product stoichiometry has not been reported. L . mesenteroides ssp cremoris (6),Leuconostoc mesenteroides ssp mesenteroides, and Leuconostoc mesenteroides ssp dextranicum are capable of utilizing acetaldehyde in both acidified and nonacidified milk cultures of 21 and 30 OC (7). It has also been found that addition of acetaldehydeto milk stimulates the growth of L. mesenteroides ssp cremoris and enhances plate counts (6). Collins and Speckman (8)have observed the same effect in using glucose-citrate broth. They have also shown a 2-fold increase in production of acetoin plus diacetyl. It has also been shown that acetaldehyde was reduced to ethanol by Leuconostoc species (8-10). The effect of acetaldehyde on growth, substrate utilization, and product formation by L. mesenteroides ssp cremoris growing in glucose and glucose plus citrate broths has not been reported in the literature and is presented here. Materials and Methods Organism and Cultivation Conditions. L. mesenteroides ssp cremoris strain 195 was obtained from Boll SA, France. The concentrated cell suspensions were kept as frozen cells at -80 "C in skim milk containing 30% (v/ v) added glycerol. The strain was subcultivated in MRS broth (11) at 30 "C statically. The ability to use citrate was tested on the medium described by Kempler and Mc Kay (12) and on the medium of Nickels and Leesment (13). For the experiments, the MRS broth was modified as follows: acetate and Tween 80 were omitted, glucose was sterilized by filtration and adjusted to 60 mmol/L, and citrate was adjusted to 10 mmol/L. The cells were grown 8756-7938/90/3006-042 1$02.50/0
@ 1990 American
Table I. Effects of Acetaldehyde on Specific Growth Rate and Biomass Formation at the Stationary Phase by L. mesen teroides ssp cremorip glucose (60 mM) plus citrate (10 mM) glucose (60 mM) -b
+
-
+
0.46 0.55 0.54 h-' 0.21 biomass, mg/mL 0.34 0.35 0.49 0.53 a Cells were grown in batch culture in modified MRS broth at initial pH 5.2 and 30 "C. b -, minus acetaldehyde; +, presence of acetaldehyde. 3.0 mM acetaldehyde from 5.6 mM initial concentration was lost by volatility at stationary phase. Pmam
at 30 "C and pH 5.2 (pH was not regulated). The initial concentration of 5.6 mM acetaldehyde was added; the amount of acetaldehydelost by volatilization was measured in uninoculated broth and used to correct the amounts produced by the culture. Inhibition of growth by acetaldehyde was observed when 20 mmol of acetaldehyde/L was added. Cell density was measured by monitoring the absorbance at 575 nm with a spectrophotometer (Novaspec 4049, LKB). The cells were removed from batch cultures by centrifugation, washed with distilled water, and centrifuged again. Dry cell weight was determined by drying the collected cells at 105 "C. A standard curve was drawn by plotting the absorbance versus dry cell weight. Cell growth was indicated by calculating the dry cell weight according to the standard curve. Analytical Methods. Cells were removed by centrifugation, and filtrates were assayed for substrates and products by HPLC. Glucose and ethanol were detected with a 156 refractive index detector (Altex); citrate, formate, acetate, and lactate were detected at 220 nm with a LC spectrophotometer (Waters, Yvelines, France) after separation of the compounds at 20 "C on a prepacked Aminex H P X 87-H column (Bio-Rad Laboratories, Richmond, CA) with a Waters 501 HPLC pump. The flow rate was 0.6 mL/min, and the mobile phase was 0.01 N H2S04. Quantification was made by integration of the area under each chromatographic peak (software:Chroma, Biosytgme, Dijon, France). In each case an external standard was used. Diacetyl, acetoin, 2,3-butylene glycol (2,3BG), ethanol, and acetaldehyde were determined by gas-
Chemical Society and American Institute
of
Chemical Engineers
Biotechnol. Prog., 1990, Vol. 6 , No. 6
422
to
0.6 5
A
4
T
2 0.4
7 h
3 3 0 i
!
69
L
0.2
0
30
10
B
I
0
$v
z
45
r6
E *
v
330
0".
1
15
C
Time (h)
Time (h)
Figure 1. Effect of acetaldehyde on fermentation,kineticsof L. mesenteroides ssp cremoris growing in modified MRS broth containing 60 mM glucose. The initial pH was 5.2 and the temperature of incubation was 30 "C. (A) Growth without acetaldehyde; (B) growth with 5.6 mM acetaldehyde initial concentration. Specific growth rate (b)(0); specific glucose consumption rate ( p ~(A); ) specific lactate production rate 401
r0.75
-
B I
n I
f
T
-9 2E
v
Y 9
3. 0
3
Time (h)
Time (h)
Figure 2. Effectof acetaldehyde on fermentation kinetics of L. mesenteroides ssp cremoris growing in modified MRS broth containing 60 mM glucose and 10 mM citrate. The initial pH was 5.2 and the temperature of incubation was 30 O C . (A) Growth without acetaldehyde; (B) growth with 5.6 mM acetaldehyde initial concentration. Specific growth rate (b) (0); specific glucose consumption rate ( p ~ (A); ) specific citrate consumption rate (pc) (0); specific lactate production rate ( p ~ ( ~0 )) . Effect of Acetaldehyde on Substrate Utilization. liquid chromatography according to the method of Thornhill and Cogan (14). The specificgrowth rate, specific glucose consumption rate, Calculation Mode. The specific growth rate ( p ) , the specific citrate consumption rate, and specific lactate , specific citspecific glucose consumption rate ( p ~ )the production rate were calculated from data obtained during rate consumption rate ( p c ) , and the specific lactate the growth of L. mesenteroides ssp cremoris. For cells ) calculated by using the formulas production rate ( p ~were growing on glucose, the addition of acetaldehyde (Figure p = dX/(X dt), p~ = dG/(X dt), pc = dC/(X dt), and FI,* 1)led to an increase in the specific growth rate, a decrease = dLa/(X dt), where X is the dry cell weight concentration, in the specific glucose consumption rate, and an increase G is the glucose concentration, C is the citrate concenin the specific lactate production rate. Their maxima tration, and La is the lactate concentration. For numerical appeared earlier for cells growing on glucose plus acetalanalysis, the software Eureka (Borland) was used. dehyde than when cells were grown on glucose alone. For cells growing on glucose plus citrate (Figure 21, the Results addition of acetaldehyde did not significantly change the Growth. Table I shows that L. mesenteroides ssp cremaxima of the specific citrate consumption rate, the moris grown in glucose medium in the presence or absence specific growth rate, and the specific glucose consumption of acetaldehyde and/or citrate showed marked differences rate. The specificlactate production rate was slightly lower in specific growth rate. Addition of acetaldehyde or cifor cells growing with acetaldehyde than for those growing trate to the glucose medium led to an increase in specific without it. But the appearance of these maxima occurred growth rate of 2.2- and 2.6-fold, respectively. No additional earlier in the presence of acetaldehyde. The curve of the stimulatory effect was observed for cells growing on glucose specificglucose consumption rate as a function of time that plus citrate with added acetaldehyde. An increase in biwas obtained in the presence of acetaldehyde is superomass was observed when citrate was added to medium containing glucose or glucose plus acetaldehyde. imposable on the specific citrate consumption rate. In the
423
Biotechnol. Frog., 1990, Vol. 6,No. 6
Table 11. Effects of Acetaldehyde on Fermentation Balances of L.mesenteroides ssp cremoris at the Stationary Phase.
i \ '
a
glucose
+
-b
glucose consumed, mM citrate consumed, mM lactate, mM ethanol, mM acetate, mM formate, mM acetaldehyde, mMe DAB, mMe 40
20
€
10
u
3
E L
2
0
30
1
I
I
1
4
Ethanol
Y V 3
3 U
e"
20
10
0 204
I
Acetate
10
0
10
20
20.4 na 19.4 18.6 6.2 0.006 0.4 0
Product Stoichiometries lactate mol/mol of glucose 0.81 0.95 0.91 ethanol, mol/mol of glucose 0.70 acetate mol/mol of glucose 0.11 0.30 na acetate, mol/mol of citrate na formate, mol/mol of glucose 0.0072 0.0029 formate, mol/mol of citrate 0.016 0.006 2XDAB, mol/mol of citrate na na molar growth yield, g/mol of 15.4 16.1 glucose
7 D(-)lactate
30
E
22.2 nad 17.9 15.6 2.4 0.16 0.7 0
30
40
50
Glucose used (mmol/l) Figure 3. Effect of acetaldehyde on ethanol, D-lactate, and acetate production and citrate utilization as a function of glucose consumption in L. mesenteroides ssp cremoris growing in MRS modified broth at pH 5.2 and 30 "C containing (0) 60 mM glucose, (m) 60 mM glucose plw 5.6 mM acetaldehyde initial concentration, (A)60 mM glucose plus 10 mM citrate, (+) 60 mM glucose plus 10 mM citrate plus 5.6 mM acetaldehyde. Arrows indicate end of citrate utilization. absence of acetaldehyde, the specific rate of citrate consumption was faster than the specific rate of glucose consumption. Product Formation. Substrates and end products were
glucose plus citrate (10 mM) -
+
48 10 37 24.1 15.1 0.2 0.5 0.0043
43.2 10 38.6 26.8 18.6 0.05 0.5 0.006
0.77 0.50 0.31 1.51 0.0042 0.02 0.00086 10.2
0.89 0.62 0.43 1.86 0.0011 0.005 0.0012 12.3
Cells were grown in batch culture at initial pH 5.2 and 30 'C in 60 mM glucose modified MRS broth in the presence or absence of 10 mM citrate. b Absence and presence of 5.6 mM acetaldehyde. Amount of residual acetaldehyde (5.6 mM acetaldehyde was added as initial concentration: 3.0 mM was lost by volatility). na, not applicable. e Sum of diacetyl,acetoin, and 2,3-butyleneglycol (DAB) multiplied by two because 1 mol of citrate gives 0.5 mol of DAB.
followed during the growth of L. mesenteroides ssp cremoris. The effects of acetaldehyde on citrate consumption and lactic acid, ethanol, and acetic acid production as a function of glucose consumption in the presence and absence of citrate are shown in Figure 3. The addition of acetaldehyde to cells growing on glucose led to an increase in the amount of lactate, ethanol, and acetate produced. When citrate was added to the glucose broth, the kinetics of productions of lactate and acetate were markedly increased and that of ethanol decreased independently of the presence of acetaldehyde. When acetaldehyde was added to medium containing glucose plus citrate, the production of ethanol and lactate were unchanged while that of acetate increased. As soon as citrate had been used, a decrease in lactate and acetate production and an increase in ethanol were observed. The effects of acetaldehyde on fermentation balance at the stationary phase in the presence and absence of citrate are shown in Table 11. In glucose broth, the yield of lactic acid, ethanol, and acetate corresponded to about 20% of the expected stoichiometry. The addition of acetaldehyde to broth containing glucose plus citrate or glucose alone led to an increase in yields of lactate, ethanol, and acetate and a weak enhancement of diacetyl, acetoin, and 2,3-butylene glycol (DAB). 77% of the excess of ethanol corresponded to the amount of ethanol coming from reduction of acetaldehyde. A decrease in the yield of formate was also observed.
Discussion It has been reported that acetaldehyde added to milk or to broth containing glucose plus citrate leads to an increase in the specific growth rate of L. mesenteroides ssp cremoris (6,8). The reason for this is that acetaldehyde, by serving as a hydrogen acceptor in the oxidation of NADH2, stimulates growth by permitting greater conversion of the aldehyde phosphate formed from carbohydrate (or from pyruvate derived from citrate) to
424
Biotechnol. Prog., 1990, Vol. 6, No. 6
acetate and ATP (8). The same stimulating effect was observed when citrate was added to glucose broth, which was explained by an increased production of ATP through the activity of acetate kinase (15,16). In our experiments, no significant stimulation of growth by addition of acetaldehyde was observed when the cells were grown in broth containing glucose plus citrate. These results contrast with those of Collins and Speckman (8). In addition, no significant increase in DAB was observed when acetaldehyde was added to broth containing glucose plus citrate. But the stimulating effect of acetaldehydeaddition to broth containing glucose alone was very important: the specific growth rate was enhanced about 2.5 times. In the presence of acetaldehyde the amount of acetate is enhanced, explaining the increase in specific growth rate as it has been explained above (8,15). In addition, in the absence of acetaldehyde the production of formate was greater than in the presence, probably because the pyruvateformate lyase pathway (17) is less advantageous than the pyruvate dehydrogenase pathway (181,which involves reduction of NAD+; the pool of NAD is maximal when acetaldehyde is added. The increase in specific growth rate was of the same order when citrate was added to broth containing glucose alone. An increase in biomass was observed when citrate was added to glucose broth but not when acetaldehyde was added. But when the cells were grown in the presence of citrate, the molar growth yield [ YX/G = biomass produced/ glucose consumed (g/mol)] was lower than Y X / Gof cells grown in glucose alone. Also, glucose is less efficient for biomass production in the presence of citrate but more efficient for the energetics pathways. Addition of acetaldehyde to broth containing glucose plus citrate led to an weak increase of Yx G. In the absence of acetaldehyde, specific citrate utihzation rate was higher than the glycolytic rate of L. mesenteroides ssp cremoris growing. These results show the important energetic role of citrate and acetaldehyde.
Literature Cited (1) Badings, H. T.; Galesloot, T. E. (1962). Studies on the flavour of different types of butter cultures with reference to the defect "yoghurt flavour" in butter. Proc. Int. Dairy Congr. 1962,16B, 199-208. (2) Harvey, R. J. Production of acetone and acetaldehyde by lactic streptococci. J. Dairy Res. 1960, 27, 41-45.
(3) Keenan, T. W.; Lindsay, R. C.; Morgan, M. E.; Day, E. A. Acetaldehyde production by single-strain lactic streptococci. J. Dairy Sci. 1966, 49, 10-14. (4) Keenan, T. W.; Lindsay, R. C. Removal of green flavor from ripened butter cultures. J. Dairy Sci. 1966, 49, 1563-1565. (5) Lindsay, R. C.; Day, E. A. Rapid quantitative method for determination of acetaldehyde in lactic starter cultures. J. Dairy Sci. 1965,48, 665-669. (6) Lindsay, R. C.; Day, E. A.; Sandine, W. E. Green flavor defect in lactic starter cultures. J. Dairy Sci. 1965, 48, 863-869. (7) Keenan, T. W.; Lindsay, R. C.; Day, E. A. Acetaldehyde utilization by Leuconostoc species. Appl. Microbiol. 1966,14 (51, 802-806. (8) Collins, E. B.; Speckman, R. A. Influence of acetaldehyde on growth and acetoin production by Leuconostoc citrovorum. J. Dairy Sci. 1974, 5 7 (12), 1427-1430. (9) Bills, D. D.; Day, E. A. Dehydrogenase activity of lactic s t r e p tococci. J. Dairy Sci. 1966, 49, 1473-1477. 10) Keenan, T. W. Production of acetic acid and other volatile compounds by Leuconostoc citrovorum and Leuconostoc dextranicum. Appl. Microbiol. 1968, 16 (12), 1881-1885. 11) De Man, J. C.; Rogosa, M.; Sharpe, M. E. A medium for the cultivation of lactobacilli. J.Appl. Bacteriol. 1980,23,30135. 12) Kempler, G. M.; McKay, L. L. Improved medium for detection of citrate-fermenting Streptococcus lactis ssp. diacetylactis. Appl. Enuiron. Microbiol. 1980, 39, 926-927. (13) Nickels, C.; Leesment, H. Method for the differentiation and qualitative determination of starter culture. J.Dairy Sci. 1964,42, 251-263. (14) Thornhill, P. J.; Cogan, T. M. Use of gas-liquid chromatography to determine the end products of growth of lactic acid bacteria. Appl. Enuiron. Microbiol. 1984, 47, 1250-1254. (15) Cogan, T. M. Co-metabolism of citrate and glucose by Leuconostoc ssp.: effects on growth, substrates, and products. J. Appl. Bacteriol. 1987, 63, 551-558. (16) Schmitt, P.; DiviBs, C.; Merlot, C. Utilization of citrate by Leuconostoc mesenteroides subsp. cremoris in continuous culture. Biotechnol. Lett. 1990, 12 (2), 127-130. (17) Thomas, T. D.; Turner, K. W.; Crow, V. L. Galactose fermentation by Streptococcus lactis and Streptococcus cremoris: Pathways, products and regulation. J.Bacteriol. 1980, 144,672-682. (18) Broome, M. C.; Thomas, M. P.; Hillier, A. J.; Jago, G. R. Pyruvate dehydrogenase activity in group N streptococci. A u t . J. Biol. Sci. 1980, 33, 15-25.
Accepted September 21, 1990. Registry No. Glucose, 50-99-7; lactic acid, 50-21-5; acetaldehyde, 75-07-0.
