JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 72, No. 4, 249-253. 1991

Isolation and Characterization of Mutants of Clostridium saccharo-

perbutylacetonicum Fermenting Bagasse Hydrolyzate
CHANG-SHIN CHIN, WAN-ZHE HWANG, AND HUNG CHAO LEE*
Institute of Food Science, National Chung Hsing University, Taichung, Taiwan, R.O.C.
Received 25 December 1990/Accepted 27 June 1991

Hydrolyzate obtained by enzymatic saccharification of bagasse was found non-fermentable into acetone-
butanol by Clostridium saccharoperbutylacetonicum. Mutant strains obtained by treating cells of the wild-type
strain with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methane sulfonate and plating on
bagasse hydrolyzate agar containing butanol, fermented the bagasse hydrolyzate directly for acetone-butanol
production. Some other morphological and physiological differences were observed between the wild-type
strain and several mutant strains. Production of total solvents from glucose, xylose, arabinose or cellobiose by
all the strains was carried out and compared.

During the past few years, acid hydrolysis and enzyma- tion or dilution and finally adjusted to pH 6.8 (7).
tic saccharification of lignocellulosic residues have both Solvent production experiments Fermentation exper-
been studied for the production of acetone-butanol. A iments were carried out in screw-capped test tubes contain-
general observation, however, has been that hydrolyzates, ing 20 ml medium or in 1 I cotton-plugged flasks contain-
whatever their origin, contain inhibitors of an undefined ing 800 ml medium, without flushing any gases.
nature blocking the fermentation. Different treatments For inoculum preparation, cultures maintained on RCM
that have been suggested for the removal of these inhibi- or PGM were inoculated into PGM in test tubes and
tors include the use of activated carbon, ion exchange re- placed in an anaerobic jar (Gaspak system) after heat
sins, water extraction and neutralization and sterilization shock treatment (100°C, 1 min). The jar was incubated at
(1-4). 30°C for 24-36 h. After germination, the cultures were fur-
In our laboratory we have achieved the production of ther inoculated into the same medium for 10 h incubation.
acetone-butanol from bagasse hydrolyzate by pretreating The latter was used as inoculum, and 5%0 inoculum was
the latter with active carbon or by direct fermentation of used in all experiments throughout this study.
the hydrolyzate with a 16-20 h culture of Clostridium sac- The glucose medium used contained (per 1 of distilled
charoperbutylacetonicum immobilized on calcium alginate water): (NH4)2SO4, 2 g; K2HPO4, 1 g; MgSO4"7HzO, 0.5 g;
(5, 6). In the present study we have found an efficient way FeSO4.7H20, 0.5 g; yeast extract, 5 g; CaCO3, 4 g and glu-
of isolating inhibitor-resistant mutants to make such hy- cose, 60 g; xylose, arabinose or cellobiose was also used to
drolyzate fermentable. replace glucose for solvent production (8).
The bagasse hydrolyzate medium (BHM) was prepared
by adding 2 g of (NH4)2SO4 and 4 g of CaCO 3 to 1 l of
MATERIALS AND METHODS
bagasse hydrolyzate either with or without pretreating
Microorganisms and maintenance C. saccharoper- with 5%o active carbon (5).
butylacetonicum ATCC 27022 and its mutants were main- All the experiments were conducted at 30°C for various
tained on reinforced clostridial medium (RCM, Oxoid) or times and samples were taken for determination of sugars,
potato glucose medium (PGM) containing (per 1) 250 g of acids and solvents.
cooked potatoes, 10 g of glucose, 2 g of ammonium sulfate Methods of mutation and isolation of mutants All
and 2 g of calcium carbonate, in test tubes. the cells used for mutation were grown on RCM to the
Preparation of bagasse hydrolyzate After 4 d of in- mid-exponential phase. Mutation experiments including
cubation of Trichoderma koningii W-10 at 30°C in a solid inoculation, plating, isolation, purification and incubation
medium containing 10 g of wheat bran and 10 ml of water were performed in an anaerobic glove box (Forma Scien-
in a 500 ml flask, 75 ml water was added to extract the en- tific) with controlled atmosphere (10% H2, 5% CO2 and
zymes by vigorously shaking several times and standing for 85O//ooN2). The cells were processed by the following 3 proce-
one hour. The crude enzymatic solution was obtained by dures.
centrifugation (7). (i) Cells of C. saccharoperbutylacetonicum were har-
Chopped bagasse was soaked in 4~0 NaOH (solid to vested and washed several times with 0.1 M citrate buffer,
alkali ratio 1 : 10) for 24 h and then washed with water and pH 6.8, and finally resuspended in the same buffer. The
finally dried at 50-60°C. Hydrolysis was conducted by a re- cell suspension (ca. 108 cells/ml) was treated with different
action mixture containing 180ml crude enzymatic solu- amounts (25, 50, 75, 100, 200 and 300ttg per ml) of N-
tion, 20 ml 0.5 M acetate buffer, pH 4.0, and 16 g bagasse methyl-N'-nitro-N-nitrosoguanidine (NTG) for 15min.
incubated at 50°C for 48 h on a reciprocal shaker. Bagasse One tenth ml of each was mixed with reinforced clostridial
hydrolyzate was obtained by centrifugation. The sugar con- agar (RCA, Oxoid) containing 1.7% butanol (v/v) for plat-
tent of the hydrolyzate was adjusted to 5-6% by concentra- ing. All the plates were incubated at 30°C for 24 h. Prelimi-
nary tests indicated that the wild-type strain did not grow
* Corresponding author. at this concentration of butanol. Colonies were selected
249
250 CHIN ET AL. J. FERMENT.BIOENG.,

and purified (9-11). (ii) Cultures of the parent strain were TABLE 1. Physiological characteristics of the wild-type and
plated on RCA or B H A (15 g of agar was added to 1 l of isolated strains
BHM) plates (0.1 ml plate -1) containing various volumes
27022 RT9 NGI4 705 NB1 NB3 EB9
(0.5-2.0%) of butanol. A crystal of N T G was added at the
center of each plate. The plates were then incubated at Catalase
30°C for 24 h. Colonies growing at b u t a n o l concentrations Gelatin liquefaction + + ÷ ÷ +
Indole production
which did not allow growth of the wild-type strain were Nitrite production
selected and purified (11). (iii) Ethyl methane sulfonate Carbohydrates
(EMS) was added to the parent strain cultures in a concen- dulcitol
traion of 0.05°//00 (v/v) and the resultant mixture incubated melezitose + + + + +
at 37°C for 1 h (10). Samples (0.1 ml plate -1) taken at erythritol + -
different intervals were mixed with RCA or BHA contain- melibiose + + + + + +
ing 1.7%o b u t a n o l (v/v) for plating. All the plates were lactose + + + + +
incubated at 30°C for 24 h. Colonies were selected and inositol + + + t + t
purified. rhammose + + + + + +
Analytic methods Culture supernatant fluids were
analyzed for volatile fermentation products using gas chro-
matography (8). Individual sugar of glucose, cellobiose, xy- in a similar m a n n e r as RT9 except that N T G was placed on
lose and arabinose was measured with dinitro-salicylic acid a B H A plate containing 1.5°//oo butanol.
reagent (12). Sugars in bagasse hydrolyzate were deter- Strain 705 was obtained by treating cells of the wild-type
mined by H P L C with a carbohydrate column ( P / N 84038 strain with EMS after 30 rain and further plating on RCA
S/N, Waters Associates, packed with Nucleosil. 5NH2) and containing 1.7%o butanol. Strain EB9 was isolated after
were eluted with CH3CN-water (70 : 30) at a flow rate of treating the cells with EMS for 20 min and further growing
1 ml per min (7). Cell growth was monitored by measuring on BHA containing 1.7% butanol.
the O.D. at 660 n m with a spectrophotometer or the dry Identification of mutant strains There were no great
weight at 105°C. differences in the size and morphology of the vegetative
Identification o f mutant strains Morphological and cells and spores between the wild-type strain and the iso-
physiological tests were carried out for identification of lated strains grown on glucose medium after an examination
wild-type and m u t a n t strains (13) (Hongo, M. et al., U. S. under a microscope. However, strain 705 formed pink-
Patent, 2,945,786, 1960). Routine tests including cultural colored surface colonies on RCA.
characteristics, cell morphology, nitrite production, indole Carbohydrates, including xylose, arabinose, glucose,
production, gelatin liquefaction, starch hydrolysis, chro- galactose, mannose, maltose, sucrose, cellobiose, man-
mogenesis and utilization of sugars and alcohols were nitol, dextrin, dextran, starch, salicin and pectin were
performed in an anaerobic jar. fermented by all the seven strains tested. Their other
physiological characteristics are shown in Table 1. The
new isolates acquired the ability to utilize some sugars
R E S U L T S A N D DISCUSSION
(melezitose, erythritol), which are not utilized by the wild-
Isolation of butanol-resistant and inhibitor-resistant mu- type strain. This might be due to mutation of the genes
tants Strain N G I 4 was obtained by treating cells of the that regulate the synthesis of enzymes for sugar utilization.
wild-type strain with 75/zg/ml N T G and further growing Strains NG14, RT9 and 705 obtained as described in Mate-
on a R C A plate containing 1.7°//00butanol. Strain RT9 was rials and Methods in an effort to select better solvent-pro-
isolated by placing one crystal of N T G on a RCA plate con- ducing strains belong to butanol-resistant mutants. Strains
taining 1 . 9 ~ butanol. Strains NB1 and NB3 were selected NB1, NB3 and EB9 thereby obtained are both butanol-

TABLE 2. Growth and solvent production by various mutants growing on a glucose medium
Fermentation products (g//)
Strain Lag period Doubling time Fementation time Yield
(h) (h) (h) Butanol Acetone Ethanol Total (%)
solvents
27022 4 6.2 70 16.07 4.01 0.97 21.05 35.08
(0.14) (0.41) (0.07)
RT9 0 1.8 47 17.01 3.92 1.62 22.55 37.58
(0.30) (0.28) (0.70)
NG14 2 3.2 47 17.63 3.35 0.00 20.98 34.97
(0.28) (0.32) (0.70)
705 0 3.4 35 17.34 4.11 1.03 22.48 37.47
(0.17) (0.03) (0.04)
NBI 4 5.1 70 15.21 3.97 1.21 20.39 33.98
(0.11) (0.07) (0.28)
NB3 4 6.4 60 15.72 5.02 2.03 22.77 37.95
(0.66) (0.01) (0.01)
EB9 4 6.1 70 15.53 4.27 2.07 21.87 36.45
(0.14) (0.06) (0.02)
All the flasks were incubated at 30°C for various hour. The sugar content of the medium was 62.1 g/L
Yield= weightof solvents
weight of sugar added
Figures in parentheses are standard deviations calculated from 6 fermentations.
VOL. 72, 1991 MUTANTS OF C. SACCHAROPERBUTYLACETONICUM 251

resistant and inhibitor-resistant mutants. selection conditions for isolating butanol-resistant and
Solvent production by mutant strains Solvent pro- inhibitor-resistant mutants. The time course of solvent pro-
duction by all the m u t a n t strains from glucose m e d i u m is duction by strain EB9 grown on a glucose medium and
given in Table 2, which reveals that strains RT9, NG14 and BHM respectively is shown in Fig. 1.
705 had a shorter lag period, doubling time and fermenta- Lower yields with all the strains in BHM are due to high
tion time but higher b u t a n o l production than the wild-type residual sugar, xylose and arabinose, left in the fermented
strain. medium.
A previous study reported by this laboratory indicated The shift from a high butanol ratio ( 5 : 1 8 : 7 7 =
that bagasse hydrolyzate contained four main sugars; E : A : B, av. ratio from Table 3) to a high acetone ratio
cellobiose, glucose, xylose and arabinose (7). The produc- (7 : 27 : 6 6 = E : A : B, av. ratio from Table 4) resulting
tion of total solvents by the m u t a n t strains from individual from the four individual sugars fermented by the seven
sugars is given in Table 3. This shows that strains NB1, strains, in contrast to BHM, could be due to relatively
NB3 and EB9 produced higher a m o u n t s of b u t a n o l from higher a m o u n t s of acetate added to the reaction mixture
xylose and arabinose than the wild-type strain. On the during hydrolysis for preparing bagasse hydrolyzate,
other hand, strains RT9, NG14 and 705 produced nearly which increases acetone formation, as shown in Fig. 1.
the same or slightly higher a m o u n t s of b u t a n o l from all the The u n k n o w n inhibitor present in the bagasse hydro-
sugars tested as compared to the parent strain. lyzate was found from the crude enzymatic solution by
Solvent p r o d u c t i o n by all the m u t a n t strains from BHM Hwang and Lee (unpublished data). It was an unidentified
is given in Table 4, which shows that strains NB1, NB3 and cyclic peptide secreted by the fungus, T. koningii W-10,
EB9 produced relatively higher a m o u n t s of b u t a n o l and during growth on a solid medium, as described under en-
total solvents directly from BHM than did the wild-type zyme preparation in Materials and Methods.
strain from the same medium pretreated with active car- The cyclic peptide inhibited the growth of various
bon. Strains N G I 4 and 705 could only ferment the pretreated Gram-positive bacteria including C. saccharoperbutyl-
BHM in a similar way to the wild-type strain. However, acetonicum, which was demonstrated by paper-disk plate
strain RT9, which possessed a partial inhibitor-resistant technique, but was not effective against Gram-negative
ability, produced nearly 90% of the total solvents directly bacteria. It could be a polypeptide antibiotic. Other
from BHM. cyclic peptides, including alamethicin, suzukacillin, tri-
The relatively higher a m o u n t s of b u t a n o l produced by chotoxins and trichorzianine, produced by members of
strains NB1, NB3 and EB9 directly from BHM as com- Trichoderma, have been reported by several workers (14-
pared to the wild-type strain correspond to our original 17). For identification of the resistancy of mutants to the

TABLE 3. Effect of carbon source on the production of total solvents by various mutants
Strain Ethanol Acetone Butanol Total solvents Solvent ratio Yield
(g//) (g//) (g//) (g//) (E : A : B) (°/60)
Cellobiose
27022 1.14 3.67 16.21 21.08 5 : 18 : 77 33.73
RT9 0.87 3.37 16.73 20.97 4 : 16 : 80 33.55
NG14 0.34 3.15 16.91 20.40 2 : 15 : 83 32.64
705 1.21 4.01 16.53 21.75 6 : 18 : 76 34.80
NB1 2.13 3.21 16.73 22.07 10 : 14 : 76 35.31
NB3 2.01 4.87 15.81 22.69 9 : 21 : 70 36.30
EB9 1.54 3.59 16.39 21.52 7 : 17 : 76 34.43
Arabinose
27022 1.01 3.39 16.74 21.14 5:16 79 33.82
RT9 1.21 3.81 16.75 21.77 6:17 77 34.83
NG14 0.01 3.37 17.21 20.59 0:16 84 32.94
705 1.34 4.12 17.03 22.49 6:18 76 35.98
NB1 1.21 3.93 17.33 22.47 5:18 77 35.95
NB3 1.97 4.39 17.31 23.67 8:19 73 37.87
EB9 1.34 3.81 16.97 22.12 6:17 77 35.39
Xylose
27022 0.69 2.07 14.24 17.00 4:12 84 27.20
RT9 0.97 3.01 15.62 19.60 5:15 80 31.36
NG14 0.03 4.03 14.34 18.40 0:22 78 29.44
705 0.72 3.17 14.97 18.86 4:17 79 30.18
NBI 1.07 3.75 15.72 20.54 5:18 77 32.86
NB3 1.45 4.27 15.83 21.55 7:20 73 34.48
EB9 1.34 2.97 15.34 19.65 7:15 78 31.44
Glucose
27022 0.97 4.01 16.07 21.05 5:19 76 33.68
RT9 1.62 3.92 17.01 22.55 7:17 76 36.08
NG14 0.00 3.35 17.63 20.98 0:16 84 33.57
705 1.03 4.11 17.34 22.48 5:18 77 35.97
NB1 1.21 3.97 15.21 20.39 6:19 75 32.62
NB3 2.03 5.02 15.72 22.77 9:22 69 36.43
EB9 2.07 4.27 15.53 21.87 9:20 71 34.99
All the test tubes were incubated at 30°C for 96 h. The sugar content of the respective medium was 62.5 g/l.
252 C H I N ET AL. J. FERMENT. BIOENG.,

T A B L E 4. Solvent production by various m u t a n t s growing on a bagasse hydrolyzate medium a

Residual sugar (g/100 ml) Final Ethanol Acetone Butanol Total Solvent Yield
Strain solvents
xylose Arab•nose (pH) (g//) (g//) (g//) (g//) ratio (%)
27022 ~ 0.60 0.39 6.35 0.62 4.98 11.25 16.85 4 : 29 : 67 27.49
(0.10) (0.06) (0.14)
RT9 ~ 0.28 0.24 6.58 0.58 6.53 12.11 19.22 3 : 34:63 31.35
(0.05) (0.34) (0.24)
NGI4 ~ 0.56 0.35 6.31 0.60 5.21 11.09 16.90 3 : 31 : 66 27.57
(0.07) (0.05) (0.22)
705 ~ 0.53 0.32 6.87 0.68 4.96 11.86 17.50 4 : 28 : 68 28.55
(0.24) (0.11) (0.13 )
NB1 0.39 0.30 6.43 3.54 2.87 12.42 18.83 19 : 14 : 66 30.72
(0.06) (0.08) (0.09)
NB3 0.35 0.25 6.35 1.02 4.32 13.62 18.96 5 : 23 : 72 30.93
(0.07) (0.23) (0.65)
EB9 0.32 0.23 6.42 2.28 5.03 12.18 19.49 12 : 26 : 62 31.79
(0.25) (0.27) (0.16)
RT9 0.58 0.41 6.50 1.58 5.12 9.82 16.52 10 : 31 : 59 26.95
(0.29) (0.32) (0.11)
The bagasse hydrolyzate contained glucose, 3.58 g; cellobiose, 0.61 g; xylose, 1.31 g; and arab•nose, 0.63 g per 100 ml. For medium prepara-
tion, ammonium sulfate, 0.2 g and calcium carbonate, 0.4 g were added.
b The bagasse hydrolyzate was pretreated with active carbon before medium preparation.
All the test tubes were incubated at 30°C for 96 h.
Figures in parentheses are standard deviations calculated from 3 fermentations.

total solvents
~ iV A B

/
/ butanol total solvents
20
O
J - - J - - - - _ _
o
a I ~•.... butanol
-- 10 t
> E 10 •~ o/OJ~
ac.,on. > • _ • / " _.0/ acetone
J . ¢ / r ~ ~jD-O"Ct- et hanoi

o% [ O.D. 6.5 4 o "~ --~,

.
~>---[3 ---~---~ ..... G - - C ] -cs 6 . 5
pH i

=~
o 4 ~~--~~ '~o "X3~:]-"
-~'~\ acetic acid
5.5
-i-

.~ ~- / ~\ residual sugar 4.5

.-~ ~x~ / utyriC acid " - ~ . . . ~ ~ residual sugar 3.5

butyric ac i d ~A~A~,~__A__ ~,~ A
|//' ~.acetic acid "~A-....~
0 30 60 gO 0 30 60
Time(h) T line(h)
FIG. 1. Time course of solvent production by strain EB9 grown on (A) glucose medium and (B) bagasse hydrolyzate medium.

u n k n o w n inhibitor, small paper disks impregnated with N T G directly on an agar plate containing butanol, but no
50/21 of the crude enzymatic solution were placed on in- butanol-resistant mutant was yielded with EMS. In our stu-
oculated R C A plates. An inhibition zone o f 15.2 and dies we have isolated mutants either with N T G or EMS. It
12.9 m m in diameter, respectively, was formed by strains may be a promising method for the isolation of mutants
27022 and RT9. N o inhibition zone was observed for fermenting hydrolyzates prepared by enzymatic hydrolysis
strains NB1, NB3 and EB9 (Hwang and Lee, unpublished of lignocellulosic residues to use N T G or EMS as a muta-
data). Therefore, strains NB1, NB3 and EB9 are likely to gen and to plate on hydrolyzate and butanol containing
be the antibiotic-resistant mutants, and strain RT9, a par- agar.
tially-resistant mutant.
Bowring and Morris (10) reported that mutagenesis of the
obligate anaerobe Clostridium acetobutylicum was best ACKNOWLEDGMENT
accomplished using EMS or N T G by the conventional This research was supported by a grant from the National Science
method of cell suspension. Hermann et al. (11) isolated Council o f Republic of China (NSC 76-0406-E005-01).
butanol-resistant mutants of C. acetobutylicum by placing
VOL 72, 1991 MUTANTS OF C. S A C C H A R O P E R B U T Y L A C E T O N I C U M 253

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