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Parameters Affecting Methanol Utilization by Yeasts
Zbl. Bakt. II. Abt. 136 (1981), 427 -432 [Microbial Chemistry Laboratory, National Research Centre, Dokki, Cairo, Egypt]
Parameters Affecting Methanol Utilization by Yeasts M. SALAH FODA and HODA G. EL-MASRY
Summary Screening of 28 yeast cultures, representing 22 species of various yeasts, with respect to their capabilities to assimilate methanol, has shown that this property was mostly found in certain species of the two genera Hansenula and Oandida. When methanol was used as a sole carbon source for a methanol-adapted strain of Hansenula polymorpha, a linear yield response could be obtained with increasing alcohol up to 2 % concentration. The amount of inoculum proved to be the decisive factor in determining a priori the ability of the organism to grow at 6 % methanol as final concentration. The optimum pH values for growth ranged between 4.5-5.5 with no growth at pH 6.5 or higher. A marked growth stimulation was obtained when the medium was supplied with phosphate up to 0.08 M as final concentration. Within the nitrogen sources tested, corn steep liquor concentrate gave the highest yield of cells. The significance of the obtained results are discussed with reference to feasibilities of application.
Zusammenfassung Unter 22 verschiedenen Hefearten waren Hansenula und Oandida in der Lage, Methanol als alleinige C-Quelle zu verwerten. Bis zu einem Alkoholgehalt von 2 % war bei H. polymorpha eine lineare Beziehung zwischen Alkoholzusatz und Wachstum zu verzeiehnen. Bei hoheren Methanolzusatzen (6 %) war die Grof3e des Inokulums entscheidend ffir eine a priori-Einechatzung moglichen Wachstums. Fur ein optimales Wachstum waren weiterhin erforderlich: ein pH-Bereich von 4,5-5.5 (nicht ::?: pH 6,5), ein Zusatz von P (0,08 M) sowie Maisquellwasser-Konzentrat.
Recently attention has been paid to studies aiming at the production of single-cell proteins, based on utilization of CI,compounds. Thus, a great deal of current research was oriented towards the adaptation of microbial capabilities of growth on this class of compounds to economically feasible processes with special reference to methane and methanol. Studies on the methane utilization were mainly restricted to certain species of bacteria (VARY and JOHNSON 1967, GLIKMANS 1976). On the other hand, a wider scope of microorganisms capable of methanol utilization belongs to bacteria (PEEL and QUAYLE 1961, GLIKMANS 1976), yeasts (VAN DIJKEN and HARDER 1974, TEZUKA et al. 1974) and lower fungi (TYE and WILLETTS 1977). The growing interest in production of single-cell protein using methanol stems from the evidence of possible deleterious effects developing in experimental animals, fed on hydrocarbon-grown microbial protein (KIHLBERG 1972). Since the methanol utilization by microorganisms is a relatively recent subject, the microbial technology, required for realizing the economic feasibility, is still lagging, probably due to the lack of adequate information regarding the growth physiology of the involved organisms. Thus, the present work was carried out to illustrate some of the parameters, related to methanol utilization by Hansenula polymorpha.
Materials and Methods Cultures were obtained from the Culture Collection of University of California (Davis, California) or from the Northern Regional Research Laboratory (Peoria, Illinois, USA) or from Institute of Fermentation, Osaka, Japan.
428
M. S.
F OD A
an d H . G.
E L- ~IASRY
Unless otherwise ind icated, Yeast Nitroge n Base (YNB) me di um (Dileo Laboratories , Detroit, Michigan, USA) was u sed t hro ughout t hese studies. To this me dium , which la ck s car bon source, pure gr ade methanol was added as sole source of carb on t o the indic ated concentrations. For quant itative physiolo gical studies, expone ntiall y gr owing cultures were us ed for in ocu lat ing 300 m l con ica l flasks, ea ch contain ing 40 - 50 m l of YNB me t h an ol li quid m edium. T he flasks were in cu b ated aerob ically on a rotary shak er at 30 °C for t he indicat ed periods of ti me . Gro wth wa s est.irnated eit her as d r y weight of cells per unit volu me of cult ure (gIl) or in t erms of optical de ns ity (O.D. ) of cult ure at 600 nm, u sing a Carl Zeiss Spe k ol color ime ter . To tal ni t rogen was determined b y t he mi cr o-Kjeldahl proce d ure (JA CO B 1958), an d t he protein cont en t was calculat ed b y m ul t ipl ying t he total nitrogen b y 6.25 . Car bohydrates wer e estimated b y t he anthr one method , whereas t he gravi metric procedure was u sed for determining t he lipids fr action, u sing Soxhlet ex t r act ion for 24 hI'S (C H Y K IN 1966) .
Results and Discussion 28 yeast cultures , belonging to 10 genera of ascosporogenous, ballisto sporogenous, and imperfect yeast groups, were tested for t heir abili t y to grow on YNB agar medium with methanol as a sole carbon source in t he range of 0.5 % t o 5 % concent ra tion. Ta ble 1. Gro wth of various yea st cult u res in Y NB agar slant s cont aini ng from 0.5 t o 5 % m eth anol as a sole carb on source. Growth wa s visually estimated after 7 days of incubation at 30 °C Yea st spec ies and code number
%
Methanol con cen t ration , m l 0.5
1
2
3
4
5
+
+
+
1. Y e a s t for mi ng a s co sp o r e s
Endomycopsis j ibul iger (lo ca l strain ) N emaiospora coryli U CD 66-37 H ans enula ano mala UC D C 317 H ans enula salurnus UCD 170 H ons enula pol ymorphfl IF 1476 H ans enul a philodendra NRRL 7209 H ansenulo polymorpha N RRL 7560 D ebaryomyces vanr ijii UCD 672 25 Pichia pin u s I F 1342 S accharomyce s j rayilis NRR L lI09 S accharomyce s cereoisiae (lo ca l st rain ) II. I m p e rf e c t y east s S porobolomuces salmo ni -color UCD 68371 T rigonopsis variabilis UCD 179 Oryptococcus laur entii UC D 48-23A Cryp tococcus laur enti i UCD 68-201 Candida u tilis val'. mayer NRltL 1094 Candida u tilis val'. thermop hile NR R L 10· 80 Candida ut ilis (lo ca l strain) Candida tropica lis UC D 60-31 Cand ida tropicalis I F 0006 Candida lipoly tica IF 0746 Candida lipolytica NRRL 1094 Cand ido. albicans IF 1060 Cand ida pelliculosa (lo cal st rain) Cand ida parap silosis U CD 61-27 Tor ulopsis glabrata IF 0622 Rhodotorula rubra U CD 68-295 Rhodotorul a 'rubra UCD C46A
+ +++ + +
+ ++ + +
+ + + +
+ + + +
+ + ++ ++
+ ++ ++
+
+
+
+
+
+
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+
0.5 1.0 2.0 3.0 4.0 5.0
Methanol co ncen t ration (% ) Dry weight
0. 32 0.27 0.09 0.16 0.07 0.12
0.01 0.02 0.01 0.02 0.01 0.03
0.04 0.06 0.04 0.07 0.04 0.08
0.11 0.10 0.03 0.05 0.02 0.04
1. 30 2 .40 0.80 0. 54 0.11 0.14
D.D.
D.D.
Dry weight
O.D.
0.43 0.8 0 0.27 0.18 0.04 0.05
Dr y we ight
1.30 2.40 3.7 0 4.4 0 3.40 2.50
D .D.
0.43 0.80 1.23 1.47 1.1 3 0.83
Dry weight
1.30 2.90 4.70 5.80 5.00 4.70
D .D.
10
0.43 0.97 1.57 1.93 1.70 1.57
Dry weight
1.40 2.80 4.80 5.30 5 .00 5.00
D.D.
11 -
0.47 0.93 1.60 1.77 1.70 1.70
Dry weight
..
~
I>:l
tf:o.
s'"
~ go
~
s g'
E?
S1
4
2
o time
8
~
l:l
£.
Incubation period (days )
Table 2. G r owth o f H an senula polymorpha 1476 in various con cen t r a t ions o f m ethanol, YNB liqu id m edium was used a nd the growth is exp ressed as D.D. 600 nm o f cu ltu re ; dry weight is ca lcula ted in mg cell sjrn l
~.
i
~
{;
(l>
8
~ El
430
M. S.
FODA
and H. G.
EL·MASRY
Table 1 shows the visual estimation of growth after 7 days of incubation at 30°C. Significant growth was detected only in certain species of Haneenula and Candida. Weak growth was observed for the mycelial yeast Endomycopsis jibuliger at the methanol concentration between 3-5 %' whereas Saccharomyces jragilis (NRRL 1109) could utilize methanol at lower concentrations only. H. polymorpha grew poorly in the liquid methanol-containing medium. Thus, the original strains were passed through a series of transfers, alternating between liquid shaking cultures and agar plates of YNB medium, all containing 6 % methanol. Within a period of 8 weeks a new strain could be recovered that gave better growth than the wild type. Its properties are shown in Table 2. Rapid growth was obtained with low concentration of alcohol. Relationship could be observed between the time required to attain the maximum yield and methanol concentration of the medium. On account of the apparent toxicity of methanol at elevated concentration (TYE and WILLETTS 1977) the experiment was carried out to investigate the possibility of overcoming this problem by increasing the initial amount of cells. Results are summarized in Table 3. Response of growth within 4 days was obtained with high inoculum concentration. On the other hand, the organism grew at a very slow rate with an inoculum size about 0.04 mg/ml and totally failed to exhibit significant growth using lower inoculum concentrations within the experimental period (10 days). Table 3. Effect of inoculum size on the growth of Honsenula polymorpha 1476 A in YNB medium 6 % methanol after different incubation periods
+
O.D. 600 nm and dry weight (mg/ml of culture) Amount of inoculum, mg dry after incubation period of (days) weightjrnl culture o time 4 7
0.165 0.083 0.042 0.021
o.ou 0.006
10
O.D.
Dry weight
O.D.
Dry weight
O.D.
Dry weight
O.D.
Dry weight
0.54 0.30 0.15 0.07 0.04 0.02
0.163 0.083 0.042 0.021 0.011 0.006
9.00 4.65 0.22 0.11 0.05 0.04
3.00 1.55 0.07 0.03 0.01 0.01
20.50 19.80 0.22 0.14 0.08 0.07
6.83 6.60 0.07 0.04 0.02 0.02
20.05 19.30 0.56 0.24 0.13 0.08
6.68 6.43 0.18 0.08 0.04 0.02
The pH dependence of the H. polymorpha growth is shown in Table 4. Table 4. Determination of optimum pH for growth of H. polymorpha 1475A on methanol as a sole source of carbon. YNB liquid medium containing 6 % methanol was used. The media were buffered at the indicated pH values using 0.08 M sodium phosphate buffer. For pH values lower than 5 only monobasic sodium phosphate salt was used with adjusting the pH with 0.1 N Hel as indicated Incubation period (days) Start 5 10 15
O.D. 600 nm of cultures buffered initially at pH 3.0
4.2
5.5
0.04 0.04 0.04 0.04 0.04 0.04 1.32 3.57 3.38 15.19 16.81 20.50
Dry weight of cells (mg{ml) of cultures buffered at pH
6.5
7.5
8.50 1 )
3.0
4.2
5.5
6.5
7.5
8.5
0.04 0.01 0.00 0.15
0.04 0.00 0.00 0.02
0.12 0.12 0.12 0.17
0.01 0.00 0.44 5.01
0.01 0.00 1.18 5.54
0.01 0.00 1.12 6.54
0.01 0.00 0.00 0.04
0.01 0.00 0.00 0.00
0.01 0.00 0.00 0.00
1) The non-inoculated medium was turbid slightly at this value of pH.
Parameters Affecting Methanol Utilization by Yeasts
431
Table 5. Influence of supplementation of YNB + 6 % methanol medium with different concentrations of phosphate (monobasic sodium salt) on growth of H. polymorpha 1476 A Incubation period (days)
a.D. 600 nm and dry weight (mgjml) of cultures supplied with phosphate concentrations (M) 0.00
0.02
0.04
0.08
0.12
0.20
0
a.D. Dry weight
0.42 0.14
0.42 0.14
0.42 0.14
0.42 0.14
0.42 0.14
0.42 0.14
4
a.D. Dry weight
0.32 0.11
0.36 0.12
0.33 0.11
0.40 0.13
0.32 0.11
0.29 0.09
7
a.D. Dry weight
1.46 0.48
2.00 0.66
1.92 0.64
6.24 2.08
0.91 0.30
0.68 0.23
10
a.D. Dry weight
10.35 3.45
13.49 4.49
15.55 5.18
20.71 6.90
11.89 3.96
8.80 2.93
14
a.D. Dry weight
8.10 2.70
9.15 3.05
9.90 3.30
11.00 3.66
10.00 3.33
9.10 3.03
The supplementation of the medium with phosphate in the range of 0.04 M to 0.08 M gave a significant increase in the maximum yield of cells, attained after 10 days of incubation, whereas at 0.2 M concentration slight inhibition was observed (Table 5). Equal amounts of nitrogen were added to the medium in the form of either inorganic salts of ammonia or nitrate or organic compounds, namely urea, peptone, or corn steep liquor concentrate. The corn steep liquor concentrate gave the highest yield of growth (Table 6). Several studies were carried out aiming at investigating the possibilities of the use of C1-compounds as the carbon source of microbial protein production. However, the commercial exploitation of these studies has not yet been materialized, due to low yield of cells produced per unit of methanol substrate (VAN DIJKEN, OTTO and HARDER 1976) and the toxicity of high concentrations of methanol to microorganisms (TYE and WILLETTS 1977). Our results indicate the possible adaptation of H. polymorpha to growth in the presence of 6 % methanol.
Table 6. Growth of Hansenula polymorpha 1476A on various nitrogen sources and 6% methanol as a carbon source. The basal medium used was supplemented with nitrogen compounds to give 0.1 % nitrogen as a final concentration in each case Incubation period (days)
o time
a.D. 600 nm of culture with the nitrogen source Ammonium Potassium nitrate sulfate
Ammonium Urea nitrate
Peptone
0.09
0.09
Corn steep liquor
0.09
0.09
3
0.52
0.48
0.56
0.63
0.51
0.52
7
2.90
0.86
1.61
1.70
0.96
16.68
0.09
0.09
9
4.70
0.66
1.21
1.95
1.28
13.21
11
1.70
0.41
1.21
4.10
0.87
8.36
432
M. S. FODA and H. G. Er..MASRY, Parameters Affecting Methanol Utilization by Yeasts
References CHYKIN, S.: Biochemistry Laboratory Techniques. John Wiley and Sons, Inc., New York 1966. GLIKMANS, G.: Processes for Single-Cell Protein Synthesis. IBP Report No. 24621 (1976). JACOB, M. B.: The Chemical Analysis of Foods and Food Products. Van Norstrand Co., Inc., New York 1958, p. 34. KIHLBERG, R.: The microbe as a source of food. Ann. Rev. Microbiol. 26 (1972), 427. PEEL, D., and QUAYLE, J. R.: Microbial growth on CI-compounds. 1. Isolation and characterization of Pseudomonas AMI. Bioohem, J. 81 (1961), 465. TEZUKA, H., NAKAHARA, T., MINODA, Y., and YAMADA, K.: Production of yeast cells from methanol. Agric. BioI. Chern. 39 (1974), 285. TYE, R., and WILLETTS, A.: Fungal growth on CI compounds. Quantitative aspects of growth of a methanol-utilizing strain of Trichoderma lignorum in batch culture. Appl, Environ. Microbiol. 33 (1977), 758. VAN DIJKEN, J. P., and HARDER, W.: Optimal conditions for enrichment and isolation of methanol assimilation yeasts. J. Gen. Microbiol. 84 (1974), 409. OTTO, R., and HARDER, W.: Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism. Arch. Mikrobiol. 111 (1976), 137. VARY, P. S., and JOHNSON, M. J.: Cell yields of bacteria grown on methane. Appl. Microbiol. 15 (1967),1473. Authors' address: Dr. MOHAMED SALAH FODA and Dr. HODA G. EL-MASRY, Microbial Chemistry Laboratory, National Research Centre, Dokki, Cairo, Egypt.
Parameters Affecting Methanol Utilization by Yeasts M. SALAH FODA and HODA G. EL-MASRY
Summary Screening of 28 yeast cultures, representing 22 species of various yeasts, with respect to their capabilities to assimilate methanol, has shown that this property was mostly found in certain species of the two genera Hansenula and Oandida. When methanol was used as a sole carbon source for a methanol-adapted strain of Hansenula polymorpha, a linear yield response could be obtained with increasing alcohol up to 2 % concentration. The amount of inoculum proved to be the decisive factor in determining a priori the ability of the organism to grow at 6 % methanol as final concentration. The optimum pH values for growth ranged between 4.5-5.5 with no growth at pH 6.5 or higher. A marked growth stimulation was obtained when the medium was supplied with phosphate up to 0.08 M as final concentration. Within the nitrogen sources tested, corn steep liquor concentrate gave the highest yield of cells. The significance of the obtained results are discussed with reference to feasibilities of application.
Zusammenfassung Unter 22 verschiedenen Hefearten waren Hansenula und Oandida in der Lage, Methanol als alleinige C-Quelle zu verwerten. Bis zu einem Alkoholgehalt von 2 % war bei H. polymorpha eine lineare Beziehung zwischen Alkoholzusatz und Wachstum zu verzeiehnen. Bei hoheren Methanolzusatzen (6 %) war die Grof3e des Inokulums entscheidend ffir eine a priori-Einechatzung moglichen Wachstums. Fur ein optimales Wachstum waren weiterhin erforderlich: ein pH-Bereich von 4,5-5.5 (nicht ::?: pH 6,5), ein Zusatz von P (0,08 M) sowie Maisquellwasser-Konzentrat.
Recently attention has been paid to studies aiming at the production of single-cell proteins, based on utilization of CI,compounds. Thus, a great deal of current research was oriented towards the adaptation of microbial capabilities of growth on this class of compounds to economically feasible processes with special reference to methane and methanol. Studies on the methane utilization were mainly restricted to certain species of bacteria (VARY and JOHNSON 1967, GLIKMANS 1976). On the other hand, a wider scope of microorganisms capable of methanol utilization belongs to bacteria (PEEL and QUAYLE 1961, GLIKMANS 1976), yeasts (VAN DIJKEN and HARDER 1974, TEZUKA et al. 1974) and lower fungi (TYE and WILLETTS 1977). The growing interest in production of single-cell protein using methanol stems from the evidence of possible deleterious effects developing in experimental animals, fed on hydrocarbon-grown microbial protein (KIHLBERG 1972). Since the methanol utilization by microorganisms is a relatively recent subject, the microbial technology, required for realizing the economic feasibility, is still lagging, probably due to the lack of adequate information regarding the growth physiology of the involved organisms. Thus, the present work was carried out to illustrate some of the parameters, related to methanol utilization by Hansenula polymorpha.
Materials and Methods Cultures were obtained from the Culture Collection of University of California (Davis, California) or from the Northern Regional Research Laboratory (Peoria, Illinois, USA) or from Institute of Fermentation, Osaka, Japan.
428
M. S.
F OD A
an d H . G.
E L- ~IASRY
Unless otherwise ind icated, Yeast Nitroge n Base (YNB) me di um (Dileo Laboratories , Detroit, Michigan, USA) was u sed t hro ughout t hese studies. To this me dium , which la ck s car bon source, pure gr ade methanol was added as sole source of carb on t o the indic ated concentrations. For quant itative physiolo gical studies, expone ntiall y gr owing cultures were us ed for in ocu lat ing 300 m l con ica l flasks, ea ch contain ing 40 - 50 m l of YNB me t h an ol li quid m edium. T he flasks were in cu b ated aerob ically on a rotary shak er at 30 °C for t he indicat ed periods of ti me . Gro wth wa s est.irnated eit her as d r y weight of cells per unit volu me of cult ure (gIl) or in t erms of optical de ns ity (O.D. ) of cult ure at 600 nm, u sing a Carl Zeiss Spe k ol color ime ter . To tal ni t rogen was determined b y t he mi cr o-Kjeldahl proce d ure (JA CO B 1958), an d t he protein cont en t was calculat ed b y m ul t ipl ying t he total nitrogen b y 6.25 . Car bohydrates wer e estimated b y t he anthr one method , whereas t he gravi metric procedure was u sed for determining t he lipids fr action, u sing Soxhlet ex t r act ion for 24 hI'S (C H Y K IN 1966) .
Results and Discussion 28 yeast cultures , belonging to 10 genera of ascosporogenous, ballisto sporogenous, and imperfect yeast groups, were tested for t heir abili t y to grow on YNB agar medium with methanol as a sole carbon source in t he range of 0.5 % t o 5 % concent ra tion. Ta ble 1. Gro wth of various yea st cult u res in Y NB agar slant s cont aini ng from 0.5 t o 5 % m eth anol as a sole carb on source. Growth wa s visually estimated after 7 days of incubation at 30 °C Yea st spec ies and code number
%
Methanol con cen t ration , m l 0.5
1
2
3
4
5
+
+
+
1. Y e a s t for mi ng a s co sp o r e s
Endomycopsis j ibul iger (lo ca l strain ) N emaiospora coryli U CD 66-37 H ans enula ano mala UC D C 317 H ans enula salurnus UCD 170 H ons enula pol ymorphfl IF 1476 H ans enul a philodendra NRRL 7209 H ansenulo polymorpha N RRL 7560 D ebaryomyces vanr ijii UCD 672 25 Pichia pin u s I F 1342 S accharomyce s j rayilis NRR L lI09 S accharomyce s cereoisiae (lo ca l st rain ) II. I m p e rf e c t y east s S porobolomuces salmo ni -color UCD 68371 T rigonopsis variabilis UCD 179 Oryptococcus laur entii UC D 48-23A Cryp tococcus laur enti i UCD 68-201 Candida u tilis val'. mayer NRltL 1094 Candida u tilis val'. thermop hile NR R L 10· 80 Candida ut ilis (lo ca l strain) Candida tropica lis UC D 60-31 Cand ida tropicalis I F 0006 Candida lipoly tica IF 0746 Candida lipolytica NRRL 1094 Cand ido. albicans IF 1060 Cand ida pelliculosa (lo cal st rain) Cand ida parap silosis U CD 61-27 Tor ulopsis glabrata IF 0622 Rhodotorula rubra U CD 68-295 Rhodotorul a 'rubra UCD C46A
+ +++ + +
+ ++ + +
+ + + +
+ + + +
+ + ++ ++
+ ++ ++
+
+
+
+
+
+
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+
0.5 1.0 2.0 3.0 4.0 5.0
Methanol co ncen t ration (% ) Dry weight
0. 32 0.27 0.09 0.16 0.07 0.12
0.01 0.02 0.01 0.02 0.01 0.03
0.04 0.06 0.04 0.07 0.04 0.08
0.11 0.10 0.03 0.05 0.02 0.04
1. 30 2 .40 0.80 0. 54 0.11 0.14
D.D.
D.D.
Dry weight
O.D.
0.43 0.8 0 0.27 0.18 0.04 0.05
Dr y we ight
1.30 2.40 3.7 0 4.4 0 3.40 2.50
D .D.
0.43 0.80 1.23 1.47 1.1 3 0.83
Dry weight
1.30 2.90 4.70 5.80 5.00 4.70
D .D.
10
0.43 0.97 1.57 1.93 1.70 1.57
Dry weight
1.40 2.80 4.80 5.30 5 .00 5.00
D.D.
11 -
0.47 0.93 1.60 1.77 1.70 1.70
Dry weight
..
~
I>:l
tf:o.
s'"
~ go
~
s g'
E?
S1
4
2
o time
8
~
l:l
£.
Incubation period (days )
Table 2. G r owth o f H an senula polymorpha 1476 in various con cen t r a t ions o f m ethanol, YNB liqu id m edium was used a nd the growth is exp ressed as D.D. 600 nm o f cu ltu re ; dry weight is ca lcula ted in mg cell sjrn l
~.
i
~
{;
(l>
8
~ El
430
M. S.
FODA
and H. G.
EL·MASRY
Table 1 shows the visual estimation of growth after 7 days of incubation at 30°C. Significant growth was detected only in certain species of Haneenula and Candida. Weak growth was observed for the mycelial yeast Endomycopsis jibuliger at the methanol concentration between 3-5 %' whereas Saccharomyces jragilis (NRRL 1109) could utilize methanol at lower concentrations only. H. polymorpha grew poorly in the liquid methanol-containing medium. Thus, the original strains were passed through a series of transfers, alternating between liquid shaking cultures and agar plates of YNB medium, all containing 6 % methanol. Within a period of 8 weeks a new strain could be recovered that gave better growth than the wild type. Its properties are shown in Table 2. Rapid growth was obtained with low concentration of alcohol. Relationship could be observed between the time required to attain the maximum yield and methanol concentration of the medium. On account of the apparent toxicity of methanol at elevated concentration (TYE and WILLETTS 1977) the experiment was carried out to investigate the possibility of overcoming this problem by increasing the initial amount of cells. Results are summarized in Table 3. Response of growth within 4 days was obtained with high inoculum concentration. On the other hand, the organism grew at a very slow rate with an inoculum size about 0.04 mg/ml and totally failed to exhibit significant growth using lower inoculum concentrations within the experimental period (10 days). Table 3. Effect of inoculum size on the growth of Honsenula polymorpha 1476 A in YNB medium 6 % methanol after different incubation periods
+
O.D. 600 nm and dry weight (mg/ml of culture) Amount of inoculum, mg dry after incubation period of (days) weightjrnl culture o time 4 7
0.165 0.083 0.042 0.021
o.ou 0.006
10
O.D.
Dry weight
O.D.
Dry weight
O.D.
Dry weight
O.D.
Dry weight
0.54 0.30 0.15 0.07 0.04 0.02
0.163 0.083 0.042 0.021 0.011 0.006
9.00 4.65 0.22 0.11 0.05 0.04
3.00 1.55 0.07 0.03 0.01 0.01
20.50 19.80 0.22 0.14 0.08 0.07
6.83 6.60 0.07 0.04 0.02 0.02
20.05 19.30 0.56 0.24 0.13 0.08
6.68 6.43 0.18 0.08 0.04 0.02
The pH dependence of the H. polymorpha growth is shown in Table 4. Table 4. Determination of optimum pH for growth of H. polymorpha 1475A on methanol as a sole source of carbon. YNB liquid medium containing 6 % methanol was used. The media were buffered at the indicated pH values using 0.08 M sodium phosphate buffer. For pH values lower than 5 only monobasic sodium phosphate salt was used with adjusting the pH with 0.1 N Hel as indicated Incubation period (days) Start 5 10 15
O.D. 600 nm of cultures buffered initially at pH 3.0
4.2
5.5
0.04 0.04 0.04 0.04 0.04 0.04 1.32 3.57 3.38 15.19 16.81 20.50
Dry weight of cells (mg{ml) of cultures buffered at pH
6.5
7.5
8.50 1 )
3.0
4.2
5.5
6.5
7.5
8.5
0.04 0.01 0.00 0.15
0.04 0.00 0.00 0.02
0.12 0.12 0.12 0.17
0.01 0.00 0.44 5.01
0.01 0.00 1.18 5.54
0.01 0.00 1.12 6.54
0.01 0.00 0.00 0.04
0.01 0.00 0.00 0.00
0.01 0.00 0.00 0.00
1) The non-inoculated medium was turbid slightly at this value of pH.
Parameters Affecting Methanol Utilization by Yeasts
431
Table 5. Influence of supplementation of YNB + 6 % methanol medium with different concentrations of phosphate (monobasic sodium salt) on growth of H. polymorpha 1476 A Incubation period (days)
a.D. 600 nm and dry weight (mgjml) of cultures supplied with phosphate concentrations (M) 0.00
0.02
0.04
0.08
0.12
0.20
0
a.D. Dry weight
0.42 0.14
0.42 0.14
0.42 0.14
0.42 0.14
0.42 0.14
0.42 0.14
4
a.D. Dry weight
0.32 0.11
0.36 0.12
0.33 0.11
0.40 0.13
0.32 0.11
0.29 0.09
7
a.D. Dry weight
1.46 0.48
2.00 0.66
1.92 0.64
6.24 2.08
0.91 0.30
0.68 0.23
10
a.D. Dry weight
10.35 3.45
13.49 4.49
15.55 5.18
20.71 6.90
11.89 3.96
8.80 2.93
14
a.D. Dry weight
8.10 2.70
9.15 3.05
9.90 3.30
11.00 3.66
10.00 3.33
9.10 3.03
The supplementation of the medium with phosphate in the range of 0.04 M to 0.08 M gave a significant increase in the maximum yield of cells, attained after 10 days of incubation, whereas at 0.2 M concentration slight inhibition was observed (Table 5). Equal amounts of nitrogen were added to the medium in the form of either inorganic salts of ammonia or nitrate or organic compounds, namely urea, peptone, or corn steep liquor concentrate. The corn steep liquor concentrate gave the highest yield of growth (Table 6). Several studies were carried out aiming at investigating the possibilities of the use of C1-compounds as the carbon source of microbial protein production. However, the commercial exploitation of these studies has not yet been materialized, due to low yield of cells produced per unit of methanol substrate (VAN DIJKEN, OTTO and HARDER 1976) and the toxicity of high concentrations of methanol to microorganisms (TYE and WILLETTS 1977). Our results indicate the possible adaptation of H. polymorpha to growth in the presence of 6 % methanol.
Table 6. Growth of Hansenula polymorpha 1476A on various nitrogen sources and 6% methanol as a carbon source. The basal medium used was supplemented with nitrogen compounds to give 0.1 % nitrogen as a final concentration in each case Incubation period (days)
o time
a.D. 600 nm of culture with the nitrogen source Ammonium Potassium nitrate sulfate
Ammonium Urea nitrate
Peptone
0.09
0.09
Corn steep liquor
0.09
0.09
3
0.52
0.48
0.56
0.63
0.51
0.52
7
2.90
0.86
1.61
1.70
0.96
16.68
0.09
0.09
9
4.70
0.66
1.21
1.95
1.28
13.21
11
1.70
0.41
1.21
4.10
0.87
8.36
432
M. S. FODA and H. G. Er..MASRY, Parameters Affecting Methanol Utilization by Yeasts
References CHYKIN, S.: Biochemistry Laboratory Techniques. John Wiley and Sons, Inc., New York 1966. GLIKMANS, G.: Processes for Single-Cell Protein Synthesis. IBP Report No. 24621 (1976). JACOB, M. B.: The Chemical Analysis of Foods and Food Products. Van Norstrand Co., Inc., New York 1958, p. 34. KIHLBERG, R.: The microbe as a source of food. Ann. Rev. Microbiol. 26 (1972), 427. PEEL, D., and QUAYLE, J. R.: Microbial growth on CI-compounds. 1. Isolation and characterization of Pseudomonas AMI. Bioohem, J. 81 (1961), 465. TEZUKA, H., NAKAHARA, T., MINODA, Y., and YAMADA, K.: Production of yeast cells from methanol. Agric. BioI. Chern. 39 (1974), 285. TYE, R., and WILLETTS, A.: Fungal growth on CI compounds. Quantitative aspects of growth of a methanol-utilizing strain of Trichoderma lignorum in batch culture. Appl, Environ. Microbiol. 33 (1977), 758. VAN DIJKEN, J. P., and HARDER, W.: Optimal conditions for enrichment and isolation of methanol assimilation yeasts. J. Gen. Microbiol. 84 (1974), 409. OTTO, R., and HARDER, W.: Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism. Arch. Mikrobiol. 111 (1976), 137. VARY, P. S., and JOHNSON, M. J.: Cell yields of bacteria grown on methane. Appl. Microbiol. 15 (1967),1473. Authors' address: Dr. MOHAMED SALAH FODA and Dr. HODA G. EL-MASRY, Microbial Chemistry Laboratory, National Research Centre, Dokki, Cairo, Egypt.