Ind. Eng. Chem. Prod. Res. Dev. 1982,21,537-539
537
Terpenoids by Microbial Fermentation Joachlm Schlndler Department of Blotechnology, Henkel KGaA, DuesseMorf, Federal Republic of Gsrmany
The ascomycete Ceratocystis variospora is known to synthesize monoterpenes such as geraniol, citronellol, nerol, linalool, and others. The terpenoids were produced with increased yields at excess supply of the carbon source. However, as soon as a critical product concentration was reached the metabolism of the strain was obviously inhibited. The suppressive influence could be avoided by adding the lipophilic adsorbent Amberlite XAD to the growing culture, thereby trapping the excreted metabolites. By this means the amount of terpenoids synthesized
was considerably raised.
The world-wide demand for flavors and fragrances is continuously increasing. Single fragrance chemicals such as citral, citronellol, geraniol, linalool, and others consumed in 1979 represented an estimated value of more than $200 million (Bruns, 1980). An annual growth rate for flavors and fragrances of more than 11% has been prognosticated for up to 1985 (Unger, 1980). Thereby natural flavors constitute much higher values than synthetic products. Since the traditional phytogenic resources cannot keep up with the market demand anymore, a search for new supplies has proved necessary. Today modern microbiology and fermentation technology permits the mass production of many valuable natural products and possibly also represents an interesting alternative for this product branch. It was a long-held opinion that terpenoids are synthesized exclusively by higher plants. Thereby it had been neglected, however, that the ability of numerous fungal species to produce flavors and fragrances had long been used as a taxonomical criterion (Badcock, 1939; Maga, 1976). However, at the beginning of the seventies, various reports appeared, especially those of Collins and Halim, which described the discovery of terpenoids in the cultures of several fungal strains. They identified such metabolites as citronellol, citronellyl acetate, geranial, geraniol, geranyl acetate, linalool, and neral in submerse cultures of the fungus Ceratocystis variospora (Collins and Halim, 1970). In the meantime, further papers have been published presenting a great variety of fungal strains producing flavors or fragrances.
Results and Discussion In search of interesting microorganisms which synthesize fragrances in significant amounts, the ascomycete C. variospora was chosen. This strain seemed to be suitable also for the cultivation in fermenter vessels since it does not lose its metabolic capabilities-unlike many other fungi-when grown under submerse conditions rather than as surface cultures. This strain catalyzes the de novo synthesis of terpenoids from carbon sources such as glucose or hydrolysed starch. It was our intention to increase the yields of these metabolites. This was possible by increasing the amount of carbon substrate in the nutrient solution with simultaneous limitation of its nitrogen content. C. variospora tolerated concentrations of hydrolyzed starch as high as 12% in shake cultures and reached its maximum of odor development at this level. A further increase of the substrate concentration did not cause an additional rise in product yield. This inhibition could either be due to intracellular 0196-4321/82/1221-0537$01.25/0
regulatory mechanisms caused by higher concentrations of the substrate and/or products or to the antifungal effect of the accumulated terpenoids themselves, or to both of them. The microbicidal effect of essential oils, also on species such as Ceratocystis, has already been published (Narazimha Rao and Joseph, 1971; Kurita et al., 1981). In order to determine if C. variospora is inhibited by its own excreted terpenoids, the oxygen respiration rate was measured in Warburg experiments. A t low concentrations geraniol was again metabolized to some extent by resting cells of the strain. But when geraniol or linalool were added in concentrations as high as 0.33% to the washed cell suspension they were not oxidized anymore but inhibited the endogenic respiration (Figure 1). Under our experimental conditions the respiration of glucose was already inhibited in the presence of 0.1% geraniol, whereas at concentrations around 0.017 % the respiration rate was stimulated (Figure 2). Therefore in usual batch cultures no significant increase of the terpenoid synthesis above the critical level can be expected. We tried to by-pass this obstacle by adding the lipophilic adsorber Amberlite XAD to the producing shake cultures. The Amberlite-welded in perlon bags-was added to the cultures in the late growth phase. The repressive effect was largely eliminated by binding the terpenoids, the yield of the volatile components being noticeably higher than in cultures without the additive (Schindler and Bruns, 1978) (Table I). In addition to the shake flask experiments, growth- and production behavior of C. variospora was also examined in 20-L fermenters. At equal sugar concentrations the fermenter with the lower nitrogen content yielded a higher amount of terpenoids than the control. The sugar was added gradually to the culture in order to avoid a possible catabolite repression (Table 11). The yield of these odorous metabolites increased to about 1.9 g/L by circulating the fermenter broth through an external vessel with Amberlite XAD 2 (Table 111). This essential oil of fungal origin is probably comparable with the two plant products citronell oil or geranium oil (Gildemeister and Hoffmann, 1956) (Table IV). According to these authors the yield of citronell oil for instance-extracted from Andropogon nardus-amounts to between 20 and 35 kg per acre per year. The annual yield of the microbial oil obtained from a 100 m3 fermenter corresponds to that which could be won from 260 to 490 acres of arable soil. The production costs should be further reduced by optimization of the fermentation conditions and by influencing the enzymatic steps leading to the wanted metab0 1982 American Chemical Society
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Table I. Synthesis of Geraniol in Shake Cultures of Ceratocystis ___- uariospora and Its Dependence upon Amberlite XAD 2 __ geraniol, mg/L relative distribution, %a _ _ _ I -
--
_ _ l _ _ _ _ l
broth 35.5 7.9
I _ _
control 6 g/L of Amberlite
Amberlite -88.5
--
linalool ____ 1.2 :< 1.0 0.1 < 1.0 0.1 0.5 0.3
2.
35.5 96.4
1 2 g/L of Amberlite
3.2
111.0
117 2
1 8 g/L of Amberlite
8.3
212.0
220.3
citronellol 15.4 5.3 12.5 1.3 2.2 4.5 8.0
nerol 4.6 2.8 5.0 2.3 2.6
geraniol 78.7 86.7 78.8 94.8 94.5 83.5 85.9
B A B A B
A
aPercentage of volatile components extracted by n-hexane. A = eluted from Amberlite XAD 2. B = extracted from culture broth. ,I
I
140 giucase i o 1 5 c j
.-a
120
...-
100
control (endogenic rcspiration I
/
80
/*.
60
* ,
~
_.,-..*
.
i
.
,_...I.
/
I
Lc..’’. __
;/:/E-
/-
120
0.80% soybean meal 0.10% NH,Cl 12.00% starch (hydr.) 0.40% soybean meal 0.05% NH,Cl 12.00% starch (hydr.) 0.80% soybean meal 0.05% NH,Cl 16.00% starch (hydr.)
...‘ ... ..-__.-..-..__..-
_._._.-.-. -./-*-
$ 0 1 substrare 20
Table 11. Terpenoids by Fermentation as a Function of the Concentration of Nitrogen and Starch _____ culture geraniol, mg/L
f o -consumption
240
360
/
.-•
-
min
Figure 1. Respiration rate in the presence of high concentrations of terpenoids.
812 928 1141
Table 111. Yields of Terpenoids by Gradual Addition of Starch and in the Presence of Amberlite XAD-2 product yields, mg/L, in dependence on incubation time
linalool citronellol nerol gerani ol I:
72 h
96 h
120 h culture
23 26 34 1241 1324
3 130 2 1090 1225
2 57 1 323 383
1 60 1 334 395
162 h Amberlite’ 46 198 28 1227 1499 1895
‘72-162 h: culture broth was pumped through a vessel of Amberlite XAD-2. Y 120
240
-
360 min
Figure 2. Respiration of glucose in the presence of geraniol.
olites. In C. moniliformis the synthesis of terpenoids follows the mevalonate pathway as is suggested by the distribution of radioactivity in geraniol after the incubation with labeled acetate, mevalonate, or leucine (Lanza and Palmer, 1977). Whether the microbial production of these special chemicals will become of industrial importance does not only depend on the costs but also on the judgement of the perfumer concerning the olfactory qualities. Chemical methods applied for the production of terpenoids have been significantly improved (Hoffmann, 1975; Bruns, 1980) and the advantages of microbial terpenoids as natural products might be exceeded by the low prices of the synthetic substances. However, other flavors and fragrances have been identified in several fungal cultures during recent years which might be attractive enough to raise broader interest (Table V) (Collins, 1976; Collins and Halim, 1972,1977;Drawert and Barton, 1978; Francke and Bruemmer, 1978; Halim and Col!ins, 1971, 1975; Halim et al., 1975; Schmitt, 1978; Tahara et al., 1973).
Table IV. Essential Oils of Plant and Microbial Origin citronell oil
palmarosa oil
fungal oil
geraniol ( 6 0 4 5 % ) geraniol ( 7 0 4 5 % ) geraniol (40-75%) and esters and esters and esters citronellol citronellol (15%) citronellol and esters and esters (10-4396’0) and esters citronellal citronellal (5-35%) (5-10%) linalool linalool (1-2%) and others nerol and others nerol (1-6%) n e r d (1-2%) and others
Materials and Methods Microorganism and Growth Conditions. The strain C. uariospora (ATCC 12866) was obtained from the American Type Culture Collection. Shake cultures were performed in 2-L Erlenmeyer flasks with 1-L nutrient solution of the following composition NaH2P04,0.05% ; K2HP04,0.18%; NH4C1, 0.05%; MgSO4.7Hz0, 0.1070; CaC12.2H20, 0.02% ; cornsteep liquor, 1.00% ; soybean meal, 0.05%; malt extract, 1.00%; starch (hydrolyzed),
Ind. Eng. Chem. Prod. Res. Dev., Vol. 21, No. 4, 1982
539
Table V. Flavors and Fragrances of Various Microorganisms flavors or fragrances honey, rose, fruity, anise coconut pe ach fruity, grassy, almond pine, rose, apple, mushroom fruity, wintergreen, rose fruity, banana, peach, pear, rose
rose, fruity fruity, rose fruity, rose fruity, jasmine
chemical structures methylphenylacetate, geraniol, citronellol, nerol 6-pentyl-2-pyrone 6 -decalactone p-methylacetophenone, p-tolyl-1-ethanol, p-tolualdehyde thujopsene, 3-octanone, 1-octen-3-01,nerolidol, p-phenylethanol methylbenzoate, methylsalicylate, p-phenylethanol 3-methylbutyl acetate, 7 -and 01 -decalactone, geraniol, citronellol, nerol, linalool, @-terpineol 6-mthyl-5-hepten-2-01acetate, linalool, citronellol, geranyl acetate, geraniol citronellol, linalool, geraniol p-phenyle thanol, furan-2-carboxylic acid cinnamic acid methyl ester
12.00% at a pH value of 6.5. The cultures were incubated at 26 "C on a rotatory shaker (Infors AG, Type LC 6,150 rpm, 120 mm). Large-scale cultivation of the fungus was carried out in 20-L fermenters (Chemap AG) with blade stirrers at 800 rpm. The air flow rate came to 1 VVM during the first 72 h of incubation and was adjusted afterward to 0.5 W M . The fermenter was inoculated with 10 vol % of a 36 h old shake culture. The basal medium had the same composition as mentioned above but contained-l6% starch. The lipophilic adsorber Amberlite XAD 2 (Serva GmbH) was regenerated with acetone, washed with aqua dest. and welded in perlon bags (80 pm mesh). Wafburg Experiments. Manometric measurements of the O2consumption were done in a Warburg apparatus (Braun Melsungen, Type 612062) containing 2.6 mL cell suspension in 0.05 M sodium phosphate buffer of pH 6.5 in the central compartement. The reaction was started by adding 0.2 mL of the aqueous substrate solution. The Warburg vessels were incubated at 27 "C and the developing COzwas trapped in 0.2 mL of 2 N KOH in the center well. Analysis of Monoterpenes. The instrument used was a Packard-Becker Model 417, equipped with a flame ionization detector and a glass capillary column (length 15 m, 0.25 mm i.d., coated with OV 1). Carrier gas was nitrogen with a flow rate of 0.8 mL/min. The oven temperature was programmed from 80 to 180 OC with 4 "C/ min.
microorganisms Trametes odorata Trichoderma viride Sporobolomyces odorus Mycoacia uda Penicillium decumbens Phellinus species Ceratocystis moniliformis
Cera tocystis virescens Kluyveromyces lactis Ascoidea hylacoeti Inocybe corydalina
Acknowledgment The GC analysis were performed by Dr. K. Bruns and Dr. K. Wollmann. The author gratefully acknowledges also their technical advice and helpful discussions. Literature Cited Badcock, E. C. Trans. Er. Mycol. SOC.1939, 2 3 , 188. Bruns, K. Parfuem. Kosmet. 1880, 67,457. Collins, R. P. Lloydia 1976, 3 9 , 20. Collins, R. P.; Halim, A. F. Lloydk 1970, 3 3 , 481. Collins, R. P.; Hallm, A. F. J . Agric. Food Chem. 1972, 2 0 , 437. Collins, R. P.; Halim, A. F. Mycologla N 7 7 , 69, 1129. Drawert, F.; Barton, H. J . Agric. Food Chem. 1978, 26, 765. Francke, W.; Bruemmer, B. Planta Med. 1978, 3 4 , 332. Gildemeister, E., Hoffmann, F. "Die aetherischen Oele"; Akademie-Verlag: Berlin, 1956; VoI. 1V. Hallm, A. F.; Collins, R. P. Lloydia 1871, 3 4 , 451. Halim, A. F.; Collins, R. P. Lloyd& 1975, 3 8 , 87. Hallm, A. F.; Narciso, J. A.; Collins, R. P. Mycologia 1875, 6 7 , 1158. Hoffmann, W. Seifen, &le, Fette, Wachse 1975, 707,89. Kurita, N.; MiyaJi,M.; Kurane, R.; Takahara, Y. Agric. Biol. Chem. 1981, 4 5 , 954. Lanza, E.; Palmer, J. K. Phyfochemistfy 1977, 76,1555. Maga, J. A. Chem. Senses Flavour 1976, 2, 255. Narazimha Rao, B. G. V.; Joseph, P. L. Rlechst. Aromen Koerperpflegem. 1971, 2 7 , 405. Schindler, J.; Bruns, K. DOS 2840 143, 1978, Henkel KGaA. Schmitl, J. A. Naturforsch. 1978, 33c, 817. Tahara, S.;Fujlwara, K.; Mlzutani, J. Agric. Bioi. Chem. 1973, 3 7 , 2855. Unger, L. Perfum. Flavor. 1980, 5 , 35.
Received for review November 2, 1981 Accepted March 17, 1982 Paper presented at the 182nd National Meeting of the American Chemical Scoiety, New York, NY, Aug 23-28,1981; Paper AGFD No. 45.
