CURRENT MICROBIOLOGY Vol. 14 (1987), pp.

295-299

Current Microbiology
9 Springer-Verlag New York Inc. 1987

Butanol Tolerance and Autobacteriocin Production by Clostridium acetobutylicum

Philippe Soucaille, Gwena~l Joliff, Anne Izard, and Gerard Goma
National Institute of Applied Sciences, Department of Biochemical Engineering, UA-CNRS 544, Toulouse, France

Abstract. Serial enrichment was used to obtain a strain of Clostridium acetobutylicum demonstrating a 40% increase in tolerance to added butanol and a further 17% improvement in the ability to produce butanol. The glucose and the butyrate consumption were higher for the G1 variant than for the wild-type strain. In consideration of the higher butanol tolerance, a disappointly low titer of butanol was produced by the new strain. This can be explained by the higher level of autobacteriocin produced by G1. Control of autobacteriocin production appears to be one of the key factors limiting the potential of the acetone butanol fermentation.
of Monot et ai. [11] at 35~ with a constant pH of 4.8 regulated by addition of liquid ammonia (12 N) and glucose (80 g 9 liter -1) as carbon source.

The production of acetone, butanol, and ethanol by Clostridium acetobutylicum is one of the oldest industrial scale fermentations, and many reviews of this process have been published [14, 17]. Current research follows four directions: development of continuous fermentation technology [4]; understanding of the physiological relationship between organic acid metabolism and solvent production [10, 19]; characterization of the inhibition by autobacteriocin [1, 16]; and the derivation of strains with improved tolerance to butanol [5, 9]. Inhibition by butanol and autobacteriocin appears to be the primary limitation in the typical butanol fermentation process utilizing C. acetobutylicum [1]. As these two products act in synergy [16], it is of particular interest to obtain strains resistant to butanol; this permits the independent study of butanol and autobacteriocin inhibition. The objective of this study was to obtain a butanol-tolerant strain from C. acetobutylicum ATCC 824 by serial subcultures into media containing increasingly higher butanol concentrations. A strain was subsequently selected and characterized in a batch fermentation to determine both its tolerance to butanol and its production of autobacteriocin. Materials and Methods
Organism and cultivation. Clostridium acetobutylicurn ATCC 824 was maintained anaerobically in the spore form by use of the medium of Petitdemange et at. [13]. Growth at 35~ for 4 days after inoculation with spores was followed by storage at -20~ Batch growth of C. acetobutylicum was in the synthetic medium

Development of butanol tolerance. The butanol-tolerant strain, was derived from C. acetobutylicum ATCC 824 by a serial enrichment procedure similar to that used by Lin and Blaschek [9]. Samples (1 ml) of exponential phase cultures of the parent strain were inoculated into Bellco tubes (20 ml aluminum sealed tubes) containing 9 ml of synthetic medium supplemented with n-butanol (5 g 9 liter-I). Growth was monitored directly in the tube by optical density measurements at 620 nm with a Turner model spectrophotometer. Cultures with an optical density of A620 = 1 were transferred sequentially to fresh media containing increasing concentrations of n-butanol.

Growth challenge studies. The effect of the fermentation products on the growth rate of C. acetobutylicum was determined by
the following procedure. A 12-h culture was used as a 10% inoculure for Bellco tubes containing 9 ml of 6% (wt/vol) glucose synthetic medium with an initial pH of 6.5 and various concentrations of n-butanol. Growth was monitored hourly until the A620 reached 0.8. Analytical methods. Cell growth was measured by optical density determinations at 620 nm against a medium water blank. The concentration of glucose was followed with an YSI 27 glucose analyzer. Production of solvents (ethanol, acetone, and butanol) and organic acids (acetic and butyric) was quantified by gas chromatography. Acidified supernatant samples (1/~1) were injected into a Perkin-Elmer Sigma 3B chromatograph equipped with a flame ionization detector. An inox column (2 m long) packed with Porapak Q (80-100 mesh) was used with nitrogen as the carrier gas. The column temperature was maintained at 210~ the injection temperature at 280~ and the detector temperature at 240~ Peak analysis was by peak area measurements as calculated by an on-line integrator relative to an isobutanol internal standard.

Address reprint requests to: Dr. P. SoucaiUe, Institut National des Sciences Appliqu6es, Departement de G6nie Biochimique et Alimentaire, UA-CNRS 544, Avenue de Rangueil, F-31077 Toulouse C6dex, France.

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CURRENT MICROBIOLOGY VOI. 14 (1987)

S(o/t 'x,,,I
"

-.--.'i~

units of

.t,,,,,

lo

~-,o

,~

2~)

WTASr

'

/4\

"

Fig. l. Effect of different butanol concentrations on growth of Clostridium acetobutylicum ATCC 824 (11) and G1 ([2): /xB//~, the ratio of the maximum specific growth rate with a B concentration of butanol on the maximum specific growth rate without butanol added is plotted in a semilogarithmic scale versus butanol concentration.

Fig. 2. Growth, products, and autobacteriocin formation of strain ATCC 824 on synthetic medium containing 8% (wt/vol) glucose. The initial pH was 5.5 and was regulated at 4.8: O, cell concentration (scale X); A, glucose (scale S); L butyrate (scale P); &, butanol (scale P); and J , autobacteriocin activity.

Quantitative determination of extracellular autobacteriocin activity was done by a method previously described [16]. Biochemical characterization of strains was accomplished with the API 20 A anaerobic diagnostic system. Calculations. Maximum specific growth rates in the presence of various concentrations of butanol were determined from the slopes of the least squares regression lines of loge (A620) vs time data.

S(o/t) Ill)3

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Results
Butanol-resistant variant selection. A butanol-resistant variant was obtained by the enrichment procedure described above. This variant, designated G l, was tolerant to butanol concentrations of 18 g 9 liter -I , i.e., was able to maintain some growth and demonstrated Gram stain reaction and microscopic morphology similar to the parent strain, although this strain had acquired raffinose, sorbitol, rhamnose, and trehalose fermenting ability. Response of Clostridium acetobutylicum to butanol. We characterized the behavior of C. acetobutylicum (wild type and G1) in response to a butanol challenge (Fig. 1). The growth rate inhibition was an exponential function of the butanol concentration, between 5 g 9 liter -I and 12 g 9 liter -I for the ATCC 824 and 8 g 9 liter -1 and 16 g 9 liter -l for G1. The specific growth rate of the parent strain was inhibited by 50% at 11.8 g 9 liter -z butanol, whereas 13.5 g 9 liter -i butanol was necessary for this degree of inhibition for G1. At a butanol concentration of 14 g liter -1 , the wild type had an apparent negative growth rate caused by cell lysis, whereas the resist-

p-6

zo

9 to

r Tlh~

Fig. 3. Growth, products, and autobacteriocin formation of strain G1 on synthetic medium containing 8% (wt/vol) glucose. The initial pH was 5.5 and was regulated at 4.8. Symbols are the same as in Fig. 1.

ant strain retained positive growth rates at butanol concentrations as high as 18 g 9 liter -l.

Solvent and autobacteriocin formation during batch culture. The progression of cell density, glucose consumption, and production of autobacteriocin and butanol were monitored during Batch culture in 8% glucose (wt/vol) synthetic medium for both wild type G1 and strain (Figs. 2 and 3 and Table 1). The maximum cell density, the final butanol and total solvents concentrations, and the maximum autobacteriocin activity were higher for the variant than for the wild-type strain. The yield o f total solvents was also better for G1 (0.31 instead of 0.276) owing

P. Soucaille et al.: Butanol Tolerance of Clostridium acetobutylicum

297

Table 1. C o m p a r i s o n of CIostridium acetobutylicum A T C C 824 and G1 during batch cultures on synthetic m e d i u m Glucose utilization (g 9 liter-0 72 -+ 1 75 -+ 1 Butanol concentration (g 9 liter ~) 12.6 - 0.2 14.7 -+ 0.2 Acetone concentration (g , liter-0 6.5 -+ 0.3 7.4 +- 0.3 Ethanol concentration (g 9 liter -1) 0.8 -+ 0.01 1.15 -+ 0.01 Final butyric acid concentration (g 9 liter -1) 1.8 -+ 0.1 0.3 --- 0.02 Final acetic acid concentration (g 9 liter -1) 1.7 --- 0.2 1.2 -+ 0.2

Strain A T C C 824 G1

Solvent yield" 0.276 0.31

a Calculated by dividing the g r a m s of solvent produced by the grams of glucose utilized.

Table 2. Concentration of principle parameters of the acetonobutylic fermentation at the point at w h i c h cell lysis begins Biomass (g 9 liter 1) Butanol (g - liter -1) Butyric acid (g - liter -~) Butanol plus butyric acid ( g . liter -~) 12 13.6 Butanol tolerance b (g - liter -1)

Strain

Autobacteriocin activity ~

A T C C 824 G1

2.66 3.4

42 62

10.5 13

1.5 0.6

13 18

E x p r e s s e d as the reciprocal of the highest dilution that did not give inhibition zone in the petri plate assay. M a x i m u m concentration of butanol that permits growth during butanol supplement challenges.

~Jfh -1)

~lp( h-t;

Fig. 4. Correlation b e t w e e n butanol produced during batch culture and the specific rate of growth of CIostridium acetobutylicum A T C C 824 (11) and G1 (D).

Fig. 5. Correlation b e t w e e n butanol produced during batch culture and the specific rate of solvent formation of Clostridium acetobutylicum A T C C 824 (A) and G1 (A).

to an improved consumption of butyric and acetic acids.

Correlation between produced butanol and the specific rates of growth and solvent production. The plot of tz versus the butanol produced (Fig. 4) for the two strains shows an apparent higher tolerance for G1 than ATCC 824. A comparison of this curve with the one of/x/~0 versus butanol added (Fig. 1) shows important differences. The growth rate of C. acetobutylicum ATCC 824 remained positive at 13 g 9 liter -] for butanol added prior to inoculation, but

for butanol or butanol plus butyric acid (Table 2) produced during growth lower maximum values (10.5 g 9 liter -t and 12 g- liter -1) were recorded. The differences were more important for G1 with 18 g 9 liter -1 for added butanol instead of 13 g 9 liter -I for produced butanol and 13.6 g 9 liter -~ for produced butanol plus butyric acid (Table 2). The specific rate of solvent production Up for the two strains is shown in Fig. 5. The maximum vp obtained for G1 and ATCC 824 was similar and achieved at the same butanol concentration of 4 g 9 liter-k The differences in the solvent production

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characteristics became apparent only when the region of each curve corresponding to inhibitory butanol concentrations was examined. Strain G1 can be seen to retain higher specific productivity values at concentrations of produced butanol which drastically cut the specific productivity values of the parent strain.

Discussion
Three mechanisms are used generally to amplify the tolerance of C. acetobutylicum to butanol: classic mutation with alkylating agents [5, 7], continuous cultivation [6], and serial transfer in batch culture with increasing concentration of butanol [9]. The G1 variant was obtained by the last method. This new strain demonstrated a 40% increase in the "static" butanol tolerance (as described above) and a further 17% improvement in the ability to produce butanol over the wild-type strain in 8% glucose synthetic medium. The isolate also acquired the ability to ferment raffinose, sorbitol, rhamnose, and trehalose, while Lin and Blaschek [9], using a similar method of selection, obtained a strain able to ferment raffinose, sorbitol, and melezitose. The acquisition of multiple fermenting ability of butanol-tolerant strains is being investigated in our laboratory in relation to a modification of the permease specificity and/or modification of the lipid bilayer composition. The inhibitory effect of straight chain alkanols, like n-butanol, is associated with fluidization of the lipid bilayer [18]. Maintenance of optimum membrane fluidity appears to be crucial to healthy cell physiology [15]. High fluidity affects membrane transport systems and their associated enzymes in C. acetobutylicum [2]. The uptake of alanine 3-0methyl glucose and membrane ATPase activity in particular are severely reduced by a butanol concentration of 10 g 9 liter i [12]. During growth in the presence of butanol or during production of butanol, C. acetobutylicum has an homeoviscous response; i.e., in response to the membrane fluidity increased by butanol, cellular fatty acid synthesis is shifted toward a higher saturated chain to unsaturated chain to decrease the membrane fluidity [18]. One possible explanation of the limiting butanol tolerance of C. acetobutylicum may be that the cell cannot sufficiently regulate its lipid composition to enable the perturbing effect of butanol to be overcome. The tolerant strain G1 obtained most probably possesses a modified lipid bilayer composition or a more rapid and effective homeoviscous re-

sponse to butanol-related membrane fluidity changes than the parent strain. It is generally admitted [8, 19] that the inhibition by butyric acid plus butanol is equivalent in term of concentration to the inhibition by butanol alone. Although some researchers [3] have demonstrated that butyric acid has a higher toxic effect than butanol, their work failed to take into account the acidification of the medium during acid supplement and the resulting pH-related inhibition of growth. The titers of butanol and butanol plus butyric acid produced by both the varianl and the wild type did not reach the levels that might be expected from the butanol supplement challenges. This was particularly true for the variant whose butanol plus butyric acid concentration, at the beginning of the lytic phase, although slightly higher than the wild type, was considerably less than the expected concentration during butanol supplements (Table 2). A plausible explanation for this disappointingly low titer is that the level of autobacteriocin for G1 was higher than the parent strain. Thus the balance of factors affecting the growth was altered, since both butanol and autobacteriocin have a synergistic effect on the lysis of C. acetobutylicum cells [16]. The mutant can be characterized as having the potential to produce significantly higher yields of butanol, but limited by the accompanying antogonistic enhancement of autobacteriocin concentration. This suggests that butanol tolerance alone will not greatly increase the biotechnological value of C. acetobutylicum. Further experimentation involving purification and characterization of the autobacteriocin as well as a genetic program to obtain autobacteriocin-negative strains will be required before the true potential of the acetone-butanol fermentation can be realized.
ACKNOWLEDGMENTS This research was supported by a grant from the Centre National de la Recherche Scientifique (CNRS-PIRSEM) and French agency of energy (AFME). We thank Dr. N.D. Lindley for assistance in the preparation of this manuscript.

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