The improvement of glucose/xylose fermentation by Clostridium acetobutylicum using calcium carbonate 

The improvement of glucose/xylose fermentation by Clostridium acetobutylicum using calcium

carbonate

A. El Kanouni,* I. Zerdani, S. Zaafa, M. Znassni, M. Lout® and M. Boudouma

Batch fermentation of 60 g/l glucose/xylose mixture by Clostridium acetobutylicum ATCC 824 was investigated on complex culture medium. Different proportions of mixtures, ranged between 10 and 50 g of each sugar/l, were fermented during pH control at 4.8 (optimum pH for solventogenesis) or during CaCO

3

addition. Using xylose- pregrown cells and pH control, an important amount of xylose was left over at the end of the fermentation when the glucose concentration was higher than that of xylose. The addition of 10 g of CaCO

3

/l (to prevent the pH dropping below 4.8) increased xylose uptake: a substantial decrease of residual xylose was observed when xylose-pregrown cells as well as glucose-pregrown cells were used as inoculum for all the mixture proportions studied. MgCO

₃

(Mg

²⁺

-containing compound) and CaCl

₂

(Ca

²⁺

-containing compound) reduced residual xylose only during pH control at 4.8 by NaOH addition. As butanol is the major limiting factor of xylose uptake in C. acetobutylicum, fermentations were carried out with or without CaCO

₃

in butanol-containing media or in iron de®cient media (under iron limitation, butanol synthesis occurred early and could inhibit xylose uptake). Results showed that an excess of CaCO

3

could increase butanol tolerance which resulted in an increase in xylose utilization. This positive effect seem to be speci®c to Ca

²⁺

- or Mg

²⁺

-containing compounds, going beyond the buffering effect of carbonate.

Key words: Calcium carbonate, Clostridium acetobutylicum, glucose/xylose fermentation.

Glucose and xylose are the more abundant sugars in the hydrolysates of hemicellulosic compounds (Dunning &

Lathrop 1945; Cowling & Kirk 1976; Donnelly et al. 1973;

Krull & Inglett 1980; Gong et al. 1981). The bioconversion of these two sugars into solvents and other chemicals is possible. However, xylose fermentation is limited by the preference of microorganisms for glucose as a carbon and energy source. Of the strict anaerobic bacteria, Clostridium acetobutylicum is one of the few microorganisms able to convert xylose and other pentoses to desirable products (ethanol, butanol and acetone) (Ounine et al. 1983), but on glucose/xylose mixtures, xylose is often left over at the end of the fermentation. Glucose-mediated catabolic re- pression and butanol inhibition of xylose transport and metabolism are the factors that limit xylose bioconversion by C. acetobutylicum (Ounine et al. 1985). To overcome this,

Maddox (1982) used co-culture of Saccharomyces cerevisiae and C. acetobutylicum. First, the yeast fermented glucose into ethanol and the residual xylose was fermented by C. acetobutylicum. However, the concentration of butanol, the most attractive end-product of this fermentation, was low. In batch fermentation, Fond et al. (1986a), used xylose-pregrown cells to show that C. acetobutylicum converted xylose when glucose was exhausted, and that glucose and xylose were converted simultaneously in fed- batch fermentations (Fond et al. 1986b). Similar results had been obtained by Groot & Luyben (1987), with an immo- bilized cell system. In both cases, a mixture of glucose and xylose was converted at low dilution rate leading to acids as major end-products. Since the hemicellulose hydrolysis may result in a mixture of

DD

-glucose and

^DD

-xylose as the major carbohydrates, it would be very important to con- vert all resulting sugars into solvents to lower the cost of the fermentation process. Xylose transport in bacteria appears to be energized by a protonmotive force (Lam et al.

1980), and high butanol concentrations decrease the membrane pH gradient due to the increase of membrane

¯uidity (Costa & Moreira 1983; Gottwald & Gotschalk

A. El Kanouni, I. Zerdani, S. Zaafa, M. Znassni and M. Boudouma are with the Faculte des Sciences Ben M'sik, DeÂpartement de Biologie, BP 7955 Laboratoire de Microbiologie, Casablanca, Morocco. M. Lout® is with the Faculte des Sci- ences Ain Choc, DeÂpartement de Biologie, Laboratoire de Biochimie et Biologie Cellulaire et MoleÂculaire, Route El Jadida, Casablanca, Morocco. *Corre- sponding author.

1985). This may result in a modi®cation of the lipid±pro- tein interactions leading to the breakdown of ATPase and permease activities during xylose fermentation by C. ace- tobutylicum. These modi®cations are pronounced in the presence of butanol and cause a drastic inhibition of xy- lose uptake. Ounine et al. (1985) found that xylose perm- ease was strongly inhibited at a butanol concentration of 8 g/l whereas glucose permease was inhibited at a buta- nol concentration of 12 g/l. In our laboratory, we ob- served that in the presence of an excess of CaCO

₃

, C. acetobutylicum exhibited best growth on xylose. This led us to study the effect of high CaCO

3

-containing medi- um on the bioconversion of glucose/xylose mixture by C. acetobutylicum and to see if this approach could be used to improve xylose utilization in glucose/xylose mixtures.

The role of Ca

²⁺

and Mg

²⁺

is discussed.

Materials and Methods

Growth Conditions for the Culture

C. acetobutylicum ATCC 824 was maintained as a spore suspen- sion in RCM (Reinforced Clostridial Medium, Oxoid) at 4

°C.

For the vegetative growing culture, spores were treated at 80

°C for 45 min and allowed to grow out in 10 ml RCM at

35

°C. For further studies, the strain was maintained by serial

transfers on xylose-containing medium to avoid degeneration.

The culture medium used as a fermentation medium contained the following (g/l): yeast extract, 4; glucose/xylose mixture, 60;

(NH

4

)

2

SO

4

, 3; K

2

HPO

4

, 0.5; and (mg/l) MgSO

4á7H2

O, 200;

MnCl

2á4H2

O, 10; (NH

4

)

6

Mo

7

O

2á4H2

O, 10; FeSO

4á7H2

O, 10. This medium, without FeSO

4

was used as iron de®ciency medium (El Kanouni et al. 1990).

Batch fermentations were performed in 150 ml ¯asks sup- plemented with CaCO

3

as buffering agent and MgCO

3

or CaCl

2

. Automatic pH control, when required, was achieved using sterilized 2

MM

NaOH. All fermentation media were autoclaved at 120

°C for 20 min. Prior to inoculation, nitrogen was bubbled

through media for 10 min.

Experimental procedure

Cell concentrations were evaluated by cell dry weight mea- surement with a predetermined correlation between absorbance at 600 nm and the cell dry weight. Concentration of residual xylose was determined by the method of Deshatelet & Yu (1986).

Total sugars was determined according to Miller et al. (1960) and the residual glucose was calculated from the difference between total sugars concentration and xylose concentration. Fermenta- tion products (ethanol, acetone, butanol, acetate and butyrate) were determined by gas chromatography using isobutanol as an internal standard (Ballongue 1984).

Results

Effect of Sugar Pregrown cells at pH 4.8 (Optimum pH for Solventogenesis)

Pro®les of the growth and sugars uptake are illustrated in Figure 1 (A and B): residual xylose at the end of

fermentation of 30 g of glucose/l and 30 g of xylose/l which was initiated with glucose-pregrown cells (Figure 1A) is higher than that initiated with xylose- pregrown cells (Figure 1B) (25 g/l and 14 g/l, respec- tively). When fermentations were carried out using var- ious glucose/xylose mixtures and xylose-pregrown cells (Figure 2A±D), no xylose uptake was observed in the mixtures in which the glucose concentration exceeded that of xylose (Figure 2A and 2B).

Effect of Calcium Carbonate, Magnesium Carbonate and Calcium Chloride

Three g/l, 6 g/l and 10 g/l of CaCO

₃

, 10 g MgCO

₃

/l and 3 g CaCl

₂

/l were supplemented to the culture me- dium containing a mixture of 30 g each sugar/l (Table 1) (concentration more than 6 g CaCl

₂

/l was toxic to the bacterium). Results indicated that 10 g calcium carbon- ate/l could improve xylose utilization without any pH control; the residual xylose was decreased from 14 to Figure 1. Growth and sugar utilization of

C. acetobutylicum

on complex medium containing 30 g of xylose/l and 30 g of glucose/l.

(A) Glucose-pregrown cells. (B) Xylose-pregrown cells.

m, glucose;

j, xylose;d, biomass.

1.8 g/l. Ten g of MgCO

₃

/l and 3 g of CaCl

₂

/l reduced residual xylose from 30 to 4.6 g/l and 2.3 g/l respec- tively, but only when the pH was adjusted using 2

MM

NaOH (MgCO

3

or CaCl

2

cannot be used as buffer compounds). Table 2 shows that for all mixtures tested,

CaCO

₃

decreased the residual xylose concentration when xylose- or glucose-adapted cells were used as inoculum.

Effect of CaCO

3

on Fermentation End-products

Results of the end-product determination are reported in Table 3. The data showed that the best solvent yield was Table 1. Effect of CaCO

3

, CaCl

2

and MgCO

3

on glucose/xylose

mixture fermentation by xylose pregrown cells of

C. acetobutyl- icum.*

Residual xylose (g/l) pH

CaCO

3

3 g/l (pH not regulated) 30 3.8

^F

CaCO

3

6 g/l (pH not regulated) 30 4.5

^F

CaCO

3

10 g/l (pH not regulated) 1.8 5.1

^F

CaCl

2

3 g/l (pH regulated) 2.3 4.8

CaCl

2

3 g/l (pH not regulated) 30 3.65

^F

MgCO

3

10 g/l (pH regulated) 4.6 4.8

MgCO

3

10 g/l (pH not regulated) 30 4

^F

Control (pH regulated)

^G

14 4.8

* Media contained 30 g of xylose/l and 30 g of glucose/l.

F

pH Measured at the end of the fermentation.

G

Fermentation control was performed using xylose-pregrown cells.

Figure 2. Growth and sugar utilization by

C. acetobutylicum

xylose-pregrown cells on complex medium containing: (A) glucose 50 g/l, xylose 10 g/l; (B) glucose 40 g/l, xylose 20 g/l; (C) glucose 10 g/l, xylose 50 g/l; (D) glucose 20 g/l, xylose 40 g/l.

m, glucose;j, xylose;d, biomass.

Table 2. Effect of CaCO

3

(10 g/l) on the fermentation of different proportions of glucose/xylose mixture by

C. acetobutylicum

(pH controlled).

Residual xylose (g/l) Sugars mixture

Glucose/xylose Glucose-pregrown

cells Xylose-pregrown cells (g/l)

10/50 6.2 4.7

20/40 4.6 3.3

30/30 2.4 1.8

40/20 2.9 2.1

50/10 2.2 1.3

3 3

obtained when the fermentation mixture was supple- mented by CaCO

₃

. In this case, 15.8 g solvents/l was accumulated (solvent/acid ratio ˆ 4.27:1) instead of 5.9 g/l (solvent/acid ratio ˆ 0.7:1) and 10.5 g/l (sol- vent/acid ratio ˆ 1.8:1) respectively in fermentations carried out at pH 4.8 using glucose- and xylose-pregrown cells.

CaCO

₃

Effect on Xylose Fermentation in the Presence of Butanol

Fermentation of 60 g xylose/l was carried out in condi- tions that allow a high butanol level in the culture me- dium by the addition of butanol, or in iron de®cient medium. When 8 g butanol/l was added to the medium at the exponential growth phase, xylose uptake could be altered and the bacterium did not use more than 30 g xylose/l during pH control. In presence of 10 g CaCO

3

/l the concentration of xylose reached 43 g/l. When C. acetobutylicum is grown in batch culture under iron limitation conditions, the carbon and electron ¯ows are altered in favour of butanol accumulation. In this con- dition, butanol was accumulated early and could inhibit xylose uptake. Results show that fermentation ceased when 16.5 g xylose/l was used and 5 g butanol/l was accumulated. The addition of 10 g CaCO

₃

/l enhanced xylose utilization which reached 41 g/l and butanol accumulation reached 9.5 g/l. Under iron limitation conditions, acid production was feeble (total acids

< 1.5 g/l), the pH did not drop below 4.8 and no pH control was required.

Discussion

C. acetobutylicum is an anaerobic bacterium which has an extended range of raw fermentable substrates. However, in batch culture medium containing glucose and xylose mixture, the bacterium metabolizes glucose ®rst and rapidly, often with incomplete xylose utilization. Our results show that the addition of an excess of CaCO

₃

in

xylose fermentation medium increases the butanol tol- erance of C. acetobutylicum and allows improved xylose utilization in spite of the persistence of glucose-mediated catabolic repression. Complete glucose/xylose mixture utilization by C. acetobutylicum was reported by Fond et al.

(1986b) during fed-batch fermentation at low substrate

¯ow. In this case, only acids were accumulated at the end of the process. Our results also show that it is possible to convert glucose/xylose mixture into solvents with good yields for all mixture proportions studied using batch fermentation modes. This phenomenon is important be- cause in a combined hemicellulosic starch hydrolysate, glucose concentration is often higher than that of xylose in the major agricultural residues (Krull & Inglett 1980).

The effect of CaCO

3

would not be due to its buffering capacity of the medium only. This is con®rmed when 8 g butanol/l was added to the culture medium. CaCO

3

addition led to the best butanol tolerance and increased xylose utilization. Similar results were observed under iron limitation: in this case, butanol was accumulated early in the fermentation (Junelles et al. 1988); the pH did not drop below 4.8 and no pH control was required.

However, an excess of CaCO

₃

addition led to good xylose utilization. These data suggest that the mechanism which increases xylose utilization may be due to the relative stability of membrane proteins provided by an excess of bivalent ions as Ca

²⁺

or Mg

²⁺

. Fermentations which were performed with MgCO

3

- or CaCl

2

-supple- mentation are in agreement with this suggestion. More performance investigations are still required to study this hypothesis.

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CaCO

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(10 g/l)

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1-Butanol 5.0 8.5 11.5

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Acetate 4.8 4.5 2.4

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(Received in revised form 5 January 1998; accepted 14 January 1998)

3 3