Folia Microbiol. 21, 409--412 (1976)
Stepwise Mutational Improvement of AspergiUus niger for Citric Acid Productivity in Cane Molasses M. A. HANNAN*, M. G. SARWAR, 2~. BATEN a n d N. CHAUDHURY
Atomic Energy Centre, Dacca, Bangladesh Received January 10, 1976
The importance of mutational improvement of industrial microorganisms has been well emphasized (Alikhanian, 1962; Calam, 1964, 1970). Variations in growth conditions as well as the composition of fermentable raw materials often warrant the selection of new strains of the organisms concerned for the desired yields (Thoma, 1971). Mutagenic techniques have been employed successfully in Aspergillus niger for enhancement of citric acid productivity (Das and Nandi, 1969; Sauchez-Marroquin et al., 1970). A number of radiation induced mutants of Aspergillus niger were found to show a remarkable increase in citric acid production in sucrose solution (Hannah et al., 1973). However, such m u t a n t s exhibited v e r y poor performance in fermentation medium prepared with cane molasses as carbon source. Our repeated experiments showed that, although mutagenic treatment of the wild type culture did produce strains with slightly higher yields of citric acid, no single t r e a t m e n t could produce a strain with an appreciable improvement of productivity in cane molasses medium. F u r t h e r attempts were therefore made to obtain such a strain through a stepwise induction of mutations contributing to higher yield. Gamma-irradiation (Co 60 source) was used for mutagenic treatments in all experiments. The wild t y p e strain, CA16, and the methods of growth and maintenance of cultures, mutagenic treatments and selection of new strains, and estimations of total sugar, total acid and citric acid in fermentation medium were the same as those described earlier (Hannah, 1972; H a n n a n et al., 1973). In the present study, however, crude cane molasses, diluted with distilled water to contain 10--14% total sugar, were used as carbon source in the fermentation medium. The inorganic salts were the same as those used b y Doelger and Prescott (1934), and the initial p H of the medium was adjusted to 3.5 with 1M HC1. Fermentation was run for 7 - - 9 days at 30 i 1 ~ and then the medium fermented by each strain was assayed. Citric acid yield was determined as percent o f the total sugar initially present in the medium (w/w). The results achieved are briefly presented in Fig. 1 showing the stepwise development of high yielding strains. It can be seen that the wild t y p e isolate, CA16 was able to show only about 15% yield of citric acid in cane molasses while the Prcse~ m~dreJs: Div. of Biological Sciences, National Research Council of Canada, Ottawa, Canada.
410
M . A . HANih;AN E T A L .
Vol. 21
IDI~] ~
NATURALISOLATE {PARENT]
5,2 C kg-I IST STEPMUTANT 2.6,5.2.7.8.10./-, C kg-I
J/
JJ /..-/
~ ' / /
N
/..
I /.,
"/~
"I/ i x .
/~
.
,
/ I /
/
~ \\
! I ~\ /
NNNNNNNN
I
~\
k
\
\\ \\~"Z.~ \ \\ ~ ~.~ ~.~ \ X "-\ \ \ ~'~
.\.~
N
N
N
3RD STEP MUTANTS (UNDER OBSERVATION]
FIa. 1. Diagram showing stepwise r~utagemc improvement of the Aapergillus niger strain CA 16 b y treatment with g a m m a irradiation. Figures in parenthesis indicate average citric acid yield (~ in relation to iugar supplied.
first-step mutant, HBs exhibited a yield around 28% under similar conditions. HB3 was found to be the best of the first-step m u t a n t s selected, and although it showed an increase in yield compared to its parent, its performance was far below what would be desired from a commercial point of view. This mutant was, therefore, exposed to gamma rays at 2.6, 5.2, 7.8 and 10.4 C k -1 and survivors were screened for improved strains. Following such treatments, a number of strains showing higher yields than HB8 were isolated and termed second-step mutants. Representatives of such m u t a n t s showing citric acid yields ranging from 39 to 71% are shown in Fig. 1. One of these mutants, numbered 79/20, which exhibited the highest yield (71%) was grown for several generations (through spore-isolation) and tested repeatedly in fermentation medium to confirm its improved productivity. This strain was then subjected to further irradiation. Our experience was that a gamma rays dose of 5.2 C kg -1 always produced the majority of the stable high yielding strains in any experiment (a typical example for the strain HBa is shown in Table I), and hence we used only this dose in subsequent experiments for inducing mutations. Following such a t r e a t m e n t of the second-step mutant, 79/20, a number of strains were obtained in which citric acid productivity was further increased. Such strains showing yields ranging from 72--92~o (Fig. 1) were termed third-step mutants, and were
1976
MUTANT SELECTION W I T H SNAIL ENZYME
411
considered for further investigations. These results, therefore, demonstrated that citric acid productivity of Aspergillus niger in cane molasses could be increased from 15 to 92% through induction of more than one m u t a t i o n in a stepwise process of selection. A comparative study of the wild type strain and the first, second and third-step mutants confirmed the marked differences in their yield potentials. It may be pointed out that we concentrated our analyses only on those strains which were found to be stable in growth as well as citric acid productivity after
TABLE I. Proportion of stable "high citric acid producing" mutants obtained from treatment with different doses of gamma rays in the strain HBz Gamma ray dose in C kg -1
Percent survival
Proportion of stable mutants per 10a survivors
0.0 2.6 5.2 7.8 10.4
100 63 43 22 11
0 1 7 3 4
repeated subcultures. We also observed that gamma-irradiation was very fruitful in producing m u t a n t s in Aspergillus niger at rather high frequencies. The results presented here indicate that citric acid productivity of Aspergillus niger in cane molasses is presumably affected by several factors which could be overcome b y gene mutations induced in the organism. This would imply t h a t multiple genes are directly or indirectly concerned with the steps/processes leading to the maximum productivity under a particular set of conditions. Under such circumstances, a single mutagenic t r e a t m e n t of a culture would probably be insufficient to produce desired strains. We, therefore, feel that in such cases a stepwise process of induction and selection of mutants would result in an accumulation of the desired mutations in one strain finally attaining the highest yield potential. Thoma (1971) presented an attractive scheme of strain improvement through the use of different mutagens in succession. Our results support this idea of stepwise improvement of industrial microorganisms to cope with the variations in fermentation conditions drastically reducing the productivity of an organism. However, we wish to point out that we achieved the desired results so far by using a single mutagen, i.e. g a m m a rays in turns.
REFERENCES ALIKHA~L~,N S. I.: Induced mutagenesis in the selection of microorganisms. Adv. Appl. Microbiol. 4, 1 (1962). C.r C. T.: The selection, improvement, and preservation of microorganisms. Progr. Ind. lllicrobiol. 5, 1 (1964). CALA~ C. T.: I m p r o v e m e n t of microorganisms by mutation, hybridization, and selection. In: Methods in Microbiology. (Norris, J. R. & Ribbons, N.W., eds.) Vol. 3A, Chap. VII, p. 435, Aead. Press, N.Y., 1970. DAs A., NANDr P.: A new strain of Aspergillus niger producing citric acid. Experientia 25, 1211 (1969). DOELGER W. P., PRESCOTT S. C.: Citric acid fermentation. Ind. Engg. Chem. 26, 1142 (1934).
412
M . A . HANNAN E T AL.
Vol. 21
H A N N A N :M. A.: Variants of Aspe~gillus niger induced by gamma rays. Ind. J. Exp. Biol. 10, 379 (1972).
HANNA-~ M. A., RABBI F., FAIZUR RAHMAN A. T. M., CHAUDHURYN.: Analysis of some mutants of Aspergillus niger for citric acid production. J. Ferment. Technol. ,~l, 606 (1973). SA~CH~.z-M)~aROQUX~ A., CAR~INO R., LEDEZ~A M.: Effect of trace elements on citric acid fermentation by Aspergillus niger. Appl. Mierobiol. 20, 888 (1970). T~o~.a R. W.: Use of mutagens in the improvement of production strains of microorganisms. Folia Mibiol. 16, 197 (1971).
Stepwise Mutational Improvement of AspergiUus niger for Citric Acid Productivity in Cane Molasses M. A. HANNAN*, M. G. SARWAR, 2~. BATEN a n d N. CHAUDHURY
Atomic Energy Centre, Dacca, Bangladesh Received January 10, 1976
The importance of mutational improvement of industrial microorganisms has been well emphasized (Alikhanian, 1962; Calam, 1964, 1970). Variations in growth conditions as well as the composition of fermentable raw materials often warrant the selection of new strains of the organisms concerned for the desired yields (Thoma, 1971). Mutagenic techniques have been employed successfully in Aspergillus niger for enhancement of citric acid productivity (Das and Nandi, 1969; Sauchez-Marroquin et al., 1970). A number of radiation induced mutants of Aspergillus niger were found to show a remarkable increase in citric acid production in sucrose solution (Hannah et al., 1973). However, such m u t a n t s exhibited v e r y poor performance in fermentation medium prepared with cane molasses as carbon source. Our repeated experiments showed that, although mutagenic treatment of the wild type culture did produce strains with slightly higher yields of citric acid, no single t r e a t m e n t could produce a strain with an appreciable improvement of productivity in cane molasses medium. F u r t h e r attempts were therefore made to obtain such a strain through a stepwise induction of mutations contributing to higher yield. Gamma-irradiation (Co 60 source) was used for mutagenic treatments in all experiments. The wild t y p e strain, CA16, and the methods of growth and maintenance of cultures, mutagenic treatments and selection of new strains, and estimations of total sugar, total acid and citric acid in fermentation medium were the same as those described earlier (Hannah, 1972; H a n n a n et al., 1973). In the present study, however, crude cane molasses, diluted with distilled water to contain 10--14% total sugar, were used as carbon source in the fermentation medium. The inorganic salts were the same as those used b y Doelger and Prescott (1934), and the initial p H of the medium was adjusted to 3.5 with 1M HC1. Fermentation was run for 7 - - 9 days at 30 i 1 ~ and then the medium fermented by each strain was assayed. Citric acid yield was determined as percent o f the total sugar initially present in the medium (w/w). The results achieved are briefly presented in Fig. 1 showing the stepwise development of high yielding strains. It can be seen that the wild t y p e isolate, CA16 was able to show only about 15% yield of citric acid in cane molasses while the Prcse~ m~dreJs: Div. of Biological Sciences, National Research Council of Canada, Ottawa, Canada.
410
M . A . HANih;AN E T A L .
Vol. 21
IDI~] ~
NATURALISOLATE {PARENT]
5,2 C kg-I IST STEPMUTANT 2.6,5.2.7.8.10./-, C kg-I
J/
JJ /..-/
~ ' / /
N
/..
I /.,
"/~
"I/ i x .
/~
.
,
/ I /
/
~ \\
! I ~\ /
NNNNNNNN
I
~\
k
\
\\ \\~"Z.~ \ \\ ~ ~.~ ~.~ \ X "-\ \ \ ~'~
.\.~
N
N
N
3RD STEP MUTANTS (UNDER OBSERVATION]
FIa. 1. Diagram showing stepwise r~utagemc improvement of the Aapergillus niger strain CA 16 b y treatment with g a m m a irradiation. Figures in parenthesis indicate average citric acid yield (~ in relation to iugar supplied.
first-step mutant, HBs exhibited a yield around 28% under similar conditions. HB3 was found to be the best of the first-step m u t a n t s selected, and although it showed an increase in yield compared to its parent, its performance was far below what would be desired from a commercial point of view. This mutant was, therefore, exposed to gamma rays at 2.6, 5.2, 7.8 and 10.4 C k -1 and survivors were screened for improved strains. Following such treatments, a number of strains showing higher yields than HB8 were isolated and termed second-step mutants. Representatives of such m u t a n t s showing citric acid yields ranging from 39 to 71% are shown in Fig. 1. One of these mutants, numbered 79/20, which exhibited the highest yield (71%) was grown for several generations (through spore-isolation) and tested repeatedly in fermentation medium to confirm its improved productivity. This strain was then subjected to further irradiation. Our experience was that a gamma rays dose of 5.2 C kg -1 always produced the majority of the stable high yielding strains in any experiment (a typical example for the strain HBa is shown in Table I), and hence we used only this dose in subsequent experiments for inducing mutations. Following such a t r e a t m e n t of the second-step mutant, 79/20, a number of strains were obtained in which citric acid productivity was further increased. Such strains showing yields ranging from 72--92~o (Fig. 1) were termed third-step mutants, and were
1976
MUTANT SELECTION W I T H SNAIL ENZYME
411
considered for further investigations. These results, therefore, demonstrated that citric acid productivity of Aspergillus niger in cane molasses could be increased from 15 to 92% through induction of more than one m u t a t i o n in a stepwise process of selection. A comparative study of the wild type strain and the first, second and third-step mutants confirmed the marked differences in their yield potentials. It may be pointed out that we concentrated our analyses only on those strains which were found to be stable in growth as well as citric acid productivity after
TABLE I. Proportion of stable "high citric acid producing" mutants obtained from treatment with different doses of gamma rays in the strain HBz Gamma ray dose in C kg -1
Percent survival
Proportion of stable mutants per 10a survivors
0.0 2.6 5.2 7.8 10.4
100 63 43 22 11
0 1 7 3 4
repeated subcultures. We also observed that gamma-irradiation was very fruitful in producing m u t a n t s in Aspergillus niger at rather high frequencies. The results presented here indicate that citric acid productivity of Aspergillus niger in cane molasses is presumably affected by several factors which could be overcome b y gene mutations induced in the organism. This would imply t h a t multiple genes are directly or indirectly concerned with the steps/processes leading to the maximum productivity under a particular set of conditions. Under such circumstances, a single mutagenic t r e a t m e n t of a culture would probably be insufficient to produce desired strains. We, therefore, feel that in such cases a stepwise process of induction and selection of mutants would result in an accumulation of the desired mutations in one strain finally attaining the highest yield potential. Thoma (1971) presented an attractive scheme of strain improvement through the use of different mutagens in succession. Our results support this idea of stepwise improvement of industrial microorganisms to cope with the variations in fermentation conditions drastically reducing the productivity of an organism. However, we wish to point out that we achieved the desired results so far by using a single mutagen, i.e. g a m m a rays in turns.
REFERENCES ALIKHA~L~,N S. I.: Induced mutagenesis in the selection of microorganisms. Adv. Appl. Microbiol. 4, 1 (1962). C.r C. T.: The selection, improvement, and preservation of microorganisms. Progr. Ind. lllicrobiol. 5, 1 (1964). CALA~ C. T.: I m p r o v e m e n t of microorganisms by mutation, hybridization, and selection. In: Methods in Microbiology. (Norris, J. R. & Ribbons, N.W., eds.) Vol. 3A, Chap. VII, p. 435, Aead. Press, N.Y., 1970. DAs A., NANDr P.: A new strain of Aspergillus niger producing citric acid. Experientia 25, 1211 (1969). DOELGER W. P., PRESCOTT S. C.: Citric acid fermentation. Ind. Engg. Chem. 26, 1142 (1934).
412
M . A . HANNAN E T AL.
Vol. 21
H A N N A N :M. A.: Variants of Aspe~gillus niger induced by gamma rays. Ind. J. Exp. Biol. 10, 379 (1972).
HANNA-~ M. A., RABBI F., FAIZUR RAHMAN A. T. M., CHAUDHURYN.: Analysis of some mutants of Aspergillus niger for citric acid production. J. Ferment. Technol. ,~l, 606 (1973). SA~CH~.z-M)~aROQUX~ A., CAR~INO R., LEDEZ~A M.: Effect of trace elements on citric acid fermentation by Aspergillus niger. Appl. Mierobiol. 20, 888 (1970). T~o~.a R. W.: Use of mutagens in the improvement of production strains of microorganisms. Folia Mibiol. 16, 197 (1971).
