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Isolation of a biodegradable sterol-rich fraction from industrial wastes
Bioresource Technology 82 (2002) 253±260
Isolation of a biodegradable sterol-rich fraction from industrial wastes A.C.P. Dias a, P. Fernandes
b,c
, J.M.S. Cabral b, H.M. Pinheiro
b,*
a Departamento de Biologia, Universidade do Minho, Campus de Gualtar, Braga 4710-057, Portugal Centro de Engenharia Biol ogica e Quõmica, Instituto Superior T ecnico, Av. Rovisco Pais, Lisboa 1049-001, Portugal Departamento de Engenharias e Tecnologias, Universidade Lus ofona de Humanidades e Tecnologias, Av do Campo Grande, Lisboa 1749-024, Portugal b
c
Received 15 September 2001; accepted 8 October 2001
Abstract Several industrial waste materials were screened for their sterol content. The possibility of using these industrial by-products as sterol sources for the microbiological production of 4-androsten-3,17-dione (AD) and 1,4-androsta-diene-3,17-dione (ADD) was investigated. Two methods of obtaining the sterol fraction from wastes were developed. Sterol-rich (96±98%) fractions were isolated in a yield above 70%, from a tall-oil euent of a paper pulp industry and from edible-oil deodorizates. These fractions were subsequently used as a substrate for microbial degradation by a Mycobacterium sp. strain and proved to be easily converted to AD and ADD. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Tall-oil; Sitosterol; Oil deodorizates; Mycobacterium sp.; Biotransformation; 4-Androstene-3,17-dione
1. Introduction Although steroid drugs represent only a small part of the world market of pharmaceuticals, there is a great demand for new and cheaper steroid raw materials for their production (Kieslich, 1985; Sedlaczek, 1988; Ahmad et al., 1992; Mahato and Garai, 1997; Angelova and Schmauder, 1999; Schmid et al., 2001). The choice of starting material has always had a critical impact on the steroid-manufacturing industries. The most common and economical process for the production of steroid pharmaceuticals is the partial synthesis from relatively inexpensive steroid raw materials of animal and plant origins. Diosgenin and other sapogenins (e.g., hecogenin) were the preferred starting materials up to the 1970s. However, the protection of Dioscorea plants reduced the availability of diosgenin and increased its price. Soybean sterols obtained from soybean oil processing were plentiful and cheap and included a large fraction of stigmasterol which can be easily converted to progesterone, thus being an excellent alternative to diosgenin (Maxon, 1985). Blunden et al. (1975) estimated that stigmasterol *
Corresponding author. Tel.: +351-1-841-9125; fax: +351-1-8419062. E-mail address: [email protected] (H.M. Pinheiro).
represented about 15% of the total precursors used in the USA, becoming, with diosgenin, one of the main raw materials for industries producing steroids of the pregnane, androstane and estrane series. Whereas stigmasterol could be degraded chemically starting with the oxidative cleavage of the 24-double bond, preserving the steroid ring structure, sterols like sitosterol and cholesterol, with saturated side-chains resistant to selective degradation, were considered low-valued or even waste products. However, since the isolation of the ®rst mutant Mycobacterium sp. strain capable of degrading the sidechain of sterols giving 17-keto-steroids (Marsheck et al., 1972) and the development of methods for the chemical addition of the corticoid side-chain to these 17-intermediates (Van Rheenen and Shephard, 1979; Neef et al., 1980), b-sitosterol, the most ubiquitous plant sterol, became a major raw material for the synthesis of corticosteroids, which represent the gross of the steroid industry (Sedlaczek, 1988). As an alternative to puri®ed sitosterol, mixed sterol concentrates obtained from natural sources such as soya (Marsheck et al., 1972) or rape seed (Schmid et al., 2001) or from industrial wastes (sugarcane and paper industries) (Srivastava et al., 1985; Perez et al., 1995; Szykula et al., 1991) have been tested as substrates for the microbial production of 17-ketosteroids. Over 1000 tons of the latter chemicals are
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 1 ) 0 0 1 8 7 - 0
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produced per year (Schmid et al., 2001). Additionally, sitosterol can be converted by other microbial mutants to 9a-hydroxy-17-keto-steroids (Sedlaczek, 1988; Seidel and H orhold, 1992) or 20-carboxy pregnane derivatives (Szentirmai, 1990) which are more suitable for corticosteroid synthesis. This paper reports on a screening of several wastes from Portuguese industries as potential sources of sterol mixtures, rich in b-sitosterol, for microbial conversion to 4-androstene-3,17-dione (AD), a convenient raw material for the steroid industry. The isolation of the sterol fraction from these wastes and the use of these sterol fractions as substrates for microbial side-chain cleavage were investigated.
2. Methods 2.1. Waste samples Tall-oil and sulfate pulp samples were a gift from Portucel ± Empresa de Celulose e Papel de Portugal SGPS, SA, from factories located at Viana do Castelo and Vila Velha do Rod~ ao, Portugal. Black liquor samples were supplied by Celulose do Caima SA (Const^ ancia, Portugal). Deodorizates from edible-oil re®ning processes were supplied by several Portuguese companies, with varied plant origins: soybean (Iberol ± Sociedade Iberica de Oleaginosas, SA, Alhandra, Portugal), sun ¯ower (Tagol ± Companhia de Oleaginosas do Tejo, SA, Almada, Portugal), peanut (Mendes Godinho, Tomar) and from a mixture of peanut and grape seed (Sovena ± Sociedade Vendedora de Glicerina, SA, Lisboa, Portugal). 2.2. Chemicals Yeast extract and potato dextrose agar (PDA) were from Difco, USA. Sterol and steroid standards were from Sigma, USA. Fructose, glycerol, chloride salts, potassium and sodium hydroxide and phosphate salts were from Merck, Germany. Ethanol, methanol, diethylether, dichloromethane, dimethyl sulfoxide, tetrahydrofurane and sulfuric and hydrochloric acids were from Riedel-de H aen, Germany. Acetone was from Fluka, Switzerland. Ammonium chloride was from J.T. Baker, The Netherlands. Hexane and ethyl acetate were from Labscan, Ireland. All these chemicals were of p.a. grade. 2.3. Production of unsaponi®ables Dried (70 °C, with vacuum, to constant weight) portions of the several waste samples were submitted to saponi®cation with a 13.6 M potassium hydroxide
solution in ethanol (1:8 v/v), in a proportion of 50 g dried waste to 112.5 ml ethanolic solution, under re¯ux, for 2±3 h. The unsaponi®able fraction was extracted three times with one volume of either diethylether or a mixture of n-hexane and ethyl acetate (5:1, v/v). The latter mixture was used when further sterol puri®cation was aimed at. The organic phase was washed with a sodium hydroxide (0.5 M) solution for the removal of acidic residues and then with water to non-alkaline reaction and dried over anhydrous sodium sulfate or calcium chloride. The organic solvent was distilled o at 40±60 °C in a rotary evaporator and the residue was dried to constant weight under vacuum at 70 °C. 2.4. Isolation of sterol-rich fractions Sterol-rich fractions were isolated from the unsaponi®able residue (above) by two distinct methods. In process P1, residue portions (5±200 g) were dissolved in 30±500 ml of petroleum ether, which was then saturated with steam to induce precipitation of the crude sterol fraction. In process P2, the residue portions (20 g) were dissolved in 100 ml of a mixture of hexane, acetone, methanol and water (49.3:34.0:15.1:1.6, v/v). The solution was then concentrated under vacuum at a temperature of 45±50 °C until sterol precipitation. In some trials, and for both processes, following sterol precipitation the remaining solution was evaporated to dryness. A second sterol-rich fraction was obtained from the recovered solid residue by repeating process P1 or P2, respectively. The crude sterol precipitates were recovered by ®ltration on ®lter paper Whatman No. 1, washed with cold (4 °C) n-hexane to remove the bulk of contaminants that precipitated together with the sterols and dried to constant weight under vacuum at 60 °C. 2.5. Biotransformation assays Biotransformations of the sterol-rich fractions isolated were carried out using Mycobacterium sp. NRRL B-3805 in 500 ml Erlenmeyer ¯asks, with a working volume of 100 ml. Bacterial cells were maintained on PDA slants (40 g l 1 ) at room temperature. Prior to fermentation cells were grown in slants for 3 days at 30 °C. The growth and bioconversion medium was composed of (in g l 1 ) fructose or glycerol (10), ammonium chloride (2), KH2 PO4 (0.75), K2 HPO4 (2.11), MgSO4 7H2 O (0.14), CaCl2 (0.025) and sterol-rich fraction (0.4 or 1.0). The inoculum was pre-cultivated in 100 ml Erlenmeyer ¯asks in a liquid medium consisting of yeast extract (10 g l 1 ) in phosphate buer 0.02 M pH 7.0, at 30 °C, with orbital shaking at 200 rpm. When the culture reached an optical density (O.D., 640 nm) of 0.6±0.9 (after 12±17 h), 2 ml were used to inoculate the larger ¯asks. Fermentations were run at 30 °C, with orbital shaking at 150 rpm and pH control at 7:0 0:3
A.C.P. Dias et al. / Bioresource Technology 82 (2002) 253±260
with 2 M NaOH or HCl solutions. Samples were collected from the liquid medium at several incubation times up to 192 h, extracted three times with ethyl acetate (1:1 v/v) and dried over anhydrous sodium sulfate or calcium chloride. The organic phase was distilled o and sterol/steroid analysis was performed by HPLC, GC, GC±MS. Cell growth was followed by measurement of protein concentration in the aqueous phase of the samples, as described by Lowry et al. (1951), after cell hydrolysis, by heating at 100 °C, for 20 min, in 1 M NaOH (Gy ure et al., 1993). 2.6. Analytical methods The presence of sterols in the samples (unsaponi®able waste raw materials, sterol-rich fractions and dried, hexane-extracted neutral fraction from mother liquors) was semi-quantitatively evaluated, after extraction with or dissolution in diethylether, by thin layer chromatography (TLC). Merck silica gel 60 plates were eluted with a mixture of dichloromethane and methanol (9:1 v/ v) and revealed with concentrated sulfuric acid in ethanol (50:50 v/v) at 100 °C. Sterol content in the unsaponi®able fractions was determined by precipitation of sterol digitonides as described elsewhere (Dias and Barroso, 1990). The sterol digitonides were cleaved with dimethyl sulfoxide as described by Issidorides et al. (1962) and the free sterols recovered. These sterols were dissolved in 200 ll tetrahydrofuran, silylated with 150 ll of hexamethyldisilazane (Merck, Germany) and 50 ll of trimethylchlorosilane (Merck, Germany) at 70 °C for 3 h, and analyzed by GC and GC±MS. Samples of the stero(l)id-rich fraction used for bioconversion were analyzed by HPLC. Quanti®cation was done by the internal standard method using progesterone as standard. Additionally, sterols and steroids were analyzed quantitatively and qualitatively by GC after trimethylsilyl derivatization. Quanti®cation was done by the internal standard method using cholestane as standard. Con®rmation of the identity of the compounds was done by GC±MS. Sterol consumption and steroid production during bioconversion were evaluated by HPLC and GC analysis. Samples were taken from the bioconversion media and extracted with two volumes of ethyl acetate. The organic phase was recovered and the solvent evaporated. The remaining stero(l)id residue was ®nally dissolved in a solution of progesterone (0:2 g l 1 ) in n-heptane (HPLC analysis) or dissolved in a solution of cholestane (0:1 g l 1 ) and silylated as described above (GC analysis). HPLC analyses were performed in a Lichrosorb Si-60 column (250 mm 40 mm; 10 lm particle diameter), with n-heptane containing 6% (v/v) ethanol as the mobile phase, at a ¯ow rate of 1:0 ml min 1 . Total sterol was detected at 215 nm and steroid products at 254 nm,
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and matched to pure standards. For total sterol, the standard was the adequate sterol-rich fraction, previously identi®ed and quanti®ed by GC±MS. GC analyses were done in a Perkin±Elmer 8600 gas chromatograph equipped with an FID detector, a datahandling system and a DB-1 fused-silica column (30 m 0:25 mm i.d., ®lm thickness 0.25 lm). The oven temperature was programmed in a ®rst slope from 200 to 220 °C, at 2 °C min 1 , and in a second from 220 to 280 °C at 5 °C min 1 , ®nally remaining at 280 °C for 30 min. Injector and detector temperatures were 300 and 320 °C, respectively. The carrier gas was H2 adjusted to a linear velocity of 300 cm s 1 . Samples were injected using the split-sampling technique with a ratio of 1:150. For GC±MS analyses the chromatograph was equipped with a DB-1 fused-silica column (30 m 0:25 mm i.d., ®lm thickness 0.25 lm) and interfaced with an iontrap detector (Finnigan Mat). Oven conditions were as described above. Transfer line and ion-trap temperatures were 280 and 220 °C, respectively. The carrier gas was He adjusted to a linear velocity of 30 cm s 1 . The split ratio was 1:40 and the ionization voltage was 70 eV, the scan range 40±500 mu and the scan time 1 s. Mass spectra and the reconstructed chromatogram were obtained by automatically scanning in the mass range m=z from 20 to 600 a.m.u. Retention time and mass spectra of the compounds in the samples were compared with those of authentic standards for identi®cation. 3. Results and discussion 3.1. Characterization of wastes in terms of sterol quality and content Earlier studies reported that tall-oil obtained as a byproduct in kraft pulp and paper mills (Conner and Rowe, 1975; Minorska and Mazgajska, 1988) as well as the wastes from the deodorization of vegetable oils (Minorska and Mazgajska, 1988) can be convenient sources of sterols, namely b-sitosterol. Therefore, several wastes from Portuguese paper pulp and edible-oil companies were examined in order to evaluate their potential importance as sterol raw material sources for the pharmaceutical industry. The results of the primary characterization of the sampled wastes are shown in Table 1. TLC analysis of all these wastes, except the black liquor from a eucalyptus pulp mill, revealed substantial amounts of sterols. The unsaponi®able matter found in Portucel tall-oil (12.7% w/w) was in agreement with the typical unsaponi®able percentages (10±40%) found in tall-oil from hardwoods (Kassebi et al., 1987). Portucel tall-oil sterol content (4.3% w/w) was similar to those found in the literature (Nekrasova et al., 1973; Conner and Rowe, 1975) and almost 10 times higher than that of Polish
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Table 1 Characteristics of examined waste samples Raw material
Water (%)
Unsaponi®able matter (%)
Sterol in unsaponi®able matter (%)
Sterol in raw material (%)
Pulp and paper Sulfate soap Tall-oil Black liquor
29 1 ngl 96 1
7.6 0.2 12.7 0.3 0.3 0.1
25 1 34 1 nd
1:4 0:2 4:3 0:2 nd
Edible-oil deodorizates Peanut/grape seed Peanut Sun¯ower Soybean
ngl ngl ngl ngl
21.1 0.2 18.0 0.3 14.3 0.2 27.1 0.3
13 2 18 1 22 0:5 20 1
2:9 0:3 3:3 0:1 3:2 0:1 5:4 0:2
All the () values were calculated on a dry weight basis and correspond to an average of at least four determinations. Sterol content was determined by the digitonine method (see materials and methods); ngl ± negligible values; nd ± sterols not detected by TLC.
tall-oil (Minorska and Mazgajska, 1988). The sterol content of tall-oil, on a dry weight basis, is nearly triple that found in sulfate soap, which is in agreement with the conversion yield obtained by Portucel (1 ton of talloil obtained from 3 tons of sulfate soap). Oil deodorizates have larger amounts of unsaponi®able matter than pulp wastes, varying from 14% to 27% (Table 1). Soy edible-oil deodorizates from Iberol also present the highest sterol content among the studied wastes. Both the unsaponi®able and sterol amounts present in deodorizate wastes are signi®cantly larger than those found in Polish deodorizates (Minorska and Mazgajska, 1988). This could be related either to dierences in the deodorization process or to the origin of the starting plant material, admitting negligible analytical discrepancies, since the methodology followed for sterol quanti®cation was very similar to the one used by the Polish authors. The unsaponi®able fractions obtained were analyzed by GC±MS and the majority of the sterols present were identi®ed (Table 2). Campesterol and b-sitosterol were present in all wastes, the latter as the main sterol present, irrespective of the waste origin. However, a signi®cant dierence in sterol composition was
observed when pulp and paper wastes or oil deodorizates were analyzed. b-Sitostanol was only detected in pulp and paper wastes, whereas stigmasterol was present in signi®cant amounts only in oil deodorizates. Sterol compositions of Portucel pulp wastes were very similar, as expected, since tall-oil is obtained from sulfate soap, and was composed mostly of b-sitosterol, in accordance with the reported sterol composition of other tall-oils (Nekrasova et al., 1973; Conner and Rowe, 1975; Szykula et al., 1991) and to that found in Portuguese Pinus pinaster whole plants which are the main wood raw materials used (results not shown). Furthermore, the processing of sulfate soap only concentrates the sterol fraction, thus not changing the relative sterol amounts. Deodorizates have sterol fractions with less b-sitosterol (46±69%) particularly the soy deodorizate (Iberol), and with much more campesterol and stigmasterol (Table 2). The presence of a relatively high content of campesterol and stigmasterol in soy deodorizates is in agreement with sterol soy oil composition which has, comparatively to peanut and sun¯ower oils, higher amounts of stigmasterol and campesterol and lower b-sitosterol amounts (Touche et al., 1975; Castang et al., 1976).
Table 2 Sterol composition of the unsaponi®able fractions of the raw materials examined Raw material
b-Sitosterol
Pulp and paper Sulfate soap Tall-oil
78 78
Oil deodorizates Peanut/grape seed Peanut Sun¯ower Soybean
62 66 69 46
Campesterol 6 5.5 12 13 13 27
Stigmasterol
b-Sitostanol
tr tr
11 11.5
12 13 14 24
nd nd nd nd
Other sterolsa 5 5 14 8 4 3
Values are expressed as the percentage by weight on the basis of the total free sterols obtained by the cleavage of the unsaponi®able fraction digitonides. The values represent the average of three independent replicates (tr ± trace amounts; nd ± not detected). a As determined by GC±MS and compared to library spectra.
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3.2. Isolation of sterol-rich fractions from wastes The use of these wastes as a source of sterols for the steroid industry required further processing of the unsaponi®able fractions in order to obtain sterol-rich fractions allowing the development of eective processes for the microbial biotransformation to corticosteroid precursors such as 4-androsten-3,17-dione (AD) and 1,4-androsten-3,17-dione (ADD). The microbial degradation of an unsaponi®able crude fraction from tall-oil led to lower bioconversion yields in AD and ADD and to a rather complex puri®cation process for the recovery of the products, as compared to the use of an upgraded substrate fraction (Szykula et al., 1991). The production of these sterol-rich fractions was achieved for deodorizates as well as for tall-oil wastes using the processes P1 and P2 described above. The results summarized in Table 3 show that in only one step, very pure sterol fractions (sterol content above 96%) could be obtained from unsaponi®able matter with high yield (above 70%). The fractions are composed of white to pale-yellow crystals with sterol compositions similar to those found in the starting unsaponi®able fractions. However some sterol remained on the mother liquors as revealed by TLC. A second precipitation of the mother liquors was carried out, using both methods, leading to second sterol-rich fractions, increasing the total sterol recovery yield up to 93% (results not shown). However these fractions were less pure, with a sterol content in the 85± 90% range and a yellow brown color. On the other hand, the use of the P1 process with tall-oil unsaponi®able fractions was found inappropriate since it gave a low purity sterol fraction in also low yield, showing that the P2 process is preferable for this kind of waste. The P2 process is a modi®cation of the protocol outlined by Johansson et al. (1977). In this process the unsaponi®able matter was ®rst dissolved in a mixture of hexane, acetone and methanol and submitted to liquid±liquid extraction of the polar compounds, including the sterols, into a hydrophilic solvent mixture, prior to crystallization of the sterol fraction, giving similar yields to those obtained in this work. Other methods referred the production of sterol-rich fractions, with a lower sterol content relatively to those obtained in the present case,
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by complexing the sterols in sulfate soap and unsaponi®able fractions from tall-oil and oil deodorizates with urea and zinc chloride, respectively (Karpus et al., 1976; Ivanov and Zlatanov, 1980). Direct supercritical carbon dioxide extraction of tall-oil seems to be selective enough to separate the neutrals, rosins and fatty acids present in tall-oil, thus isolation and puri®cation of more valuable components, e.g., b-sitosterol and squalene from neutrals, may be possible (Harvala et al., 1987). Supercritical extraction of the Portucel tall-oil unsaponi®able fraction was attempted, aimed at obtaining a sitosterol-rich fraction. Although an increased sitosterol concentration was measured in the extract, the concentration values were much lower than those obtained with the P2 process (Coelho et al., 1992). Nevertheless a previous increase in sterol concentration through supercritical extraction could help in the subsequent production of a sterol-rich fraction by the P2 process. 3.3. Bioconversion of the sterol-rich fractions One of the possible interesting uses of the sterol-rich fractions obtained is the microbial side-chain degradation to 17-keto-compounds like AD or ADD. AD and ADD are currently used as starting materials for the synthesis of corticosteroids and 19-nor-steroids, respectively (Kieslich, 1985; Ahmad et al., 1992). Sterol-rich fractions isolated from Polish tall-oil with a sterol content of approximately 20% and 84% were successfully converted by Mycobacterium sp. MB-3683 (Szykula et al., 1991). The sterol-rich fractions isolated from tall-oil unsaponi®ables and sun¯ower oil deodorizates were used in biotransformation runs with Mycobacterium sp. NRRL B-3805, a microorganism that degrades sterols to AD (the major product) and ADD (Marsheck et al., 1972). The microbial strain eectively cleaved the side-chain of the isolated sterols to AD and ADD, particularly when talloil unsaponi®able isolates were used (Table 4). The lower product yield obtained with oil deodorizate isolates could be related to their relative sterol composition. Large amounts of stigmasterol, which has a D22 -double bond in the side-chain, can be found in these deodorizates. This
Table 3 Yield and sterol content of the sterol-rich fractions obtained from the unsaponi®able fraction of tall-oil and oil deodorizate wastes, by the isolation processes described in materials and methods
a
Raw material
Isolation process
Sterol-rich fraction recovery yielda (%)
Sterol contentb (%)
Tall-oil Sun¯ower Soybean
P2 P1 P1
72 2 75 2 72 2
>96 >96 >96
Values on the basis of the total sterol amounts present in the unsaponi®able fractions (determined by the digitonine method). The values represent the minor and major values obtained from four independent replicates. b Sterol content of the isolated sterol-rich fraction on a dry weight basis (GC method), obtained in three independent isolation experiments.
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Table 4 Bioconversion and product yields of the microbial degradation of sterol-rich fractions isolated from tall-oil and sun¯ower oil processing wastes Sterol-rich fraction
Sterol-rich fraction concentration (mM)
Carbon source
Final conversion yielda (%)
Final product yieldb (%)
Sun¯ower Tall-oil Tall-oil Tall-oil
1.0 1.0 2.5 2.5
Fructose Fructose Glycerol Fructose
52 5 65 4 52 5 42 1
41 1 55 2 45 2 16 1
Values are averages from at least two independent bioconversion runs. a Calculated from sterol consumption on a molar basis. b Calculated from AD+ADD production from the initial sterol, on a molar basis.
double bond renders microbial degradation more dicult in a general way (Nagasawa et al., 1970) and the conversion of stigmasterol to androstanes by Mycobacterium sp. NRRL B-3805 has been reported to be lower than that observed with sitosterol (Marsheck et al., 1972). Indeed we observed a lower degree of conversion for stigmasterol than that observed for b-sitosterol (Fig. 1). Therefore, tall-oil wastes are the best choice for obtaining sterol-rich fractions for further bioconversions since all the main sterols present in the isolates are easily transformed. In order to enhance the process productivity, initial substrate concentrations were increased from 1.0 to 2.5 mM. Similar product yields could be obtained, as long as glycerol replaced fructose as carbon source. Possibly dierent carbon sources induce dierent metabolic activities or lead to dierent cell envelope characteristics, interfering with substrate mass transfer. Further studies are required for process optimization. Figs. 2 and 3 show a typical time course of the degradation of the sterol fractions isolated from tall-oil and sun¯ower oil deodorizates. The results in Fig. 2 con®rm that the main sterols that compose these fractions, respectively, b-sitosterol, b-sitostanol and campesterol, were metabolizable and led to AD/ADD accumulation, as veri®ed by HPLC and GC±MS analysis.
Fig. 2. Bioconversion of the sterol-rich fraction from the unsaponi®ables of tall-oil, using Mycobacterium sp. NRRL B-3805, with fructose as carbon source. b-Sitosterol (N), campesterol (d), b-sitostanol (j), AD ( ) and ADD () concentrations are shown.
Fig. 3. Bioconversion of the sterol-rich fraction from sun¯ower deodorizates, using Mycobacterium sp. NRRL B-3805, with fructose as carbon source. Overall sterol concentration (j), AD ( ) and ADD () concentrations are shown.
Fig. 1. Bioconversion of the sterol-rich fraction from the unsaponi®ables of tall-oil, using Mycobacterium sp. NRRL B-3805, with glycerol as carbon source. b-Sitosterol (N), campesterol (d), b-sitostanol (j), AD ( ), and ADD () concentrations are shown.
In order to improve the bioconversion yields of sterol-rich fractions isolated from oil deodorizates, particularly soy oil, a liquid±liquid extraction step to recover stigmasterol could be introduced. The stigmasterol thus obtained could be converted by other processes to obtain precursors for the steroid industry (Maxon, 1985) or for the synthesis of cytotoxic 7-hy-
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droxy sterols (Heltzel et al., 1994). Alternatively, the use of strains such as Mycobacterium EX-4, a mutant from Mycobacterium sp. B-3683 showing a high ability for the bioconversion of stigmasterol (Perez et al., 1995), could be preferred for the biodegradation of sterol-rich fractions from oil deodorizates.
4. Conclusion The analysis of the dierent industrial wastes considered in this study has shown that deodorizates from edible-oil re®ning and sulfate soap or tall-oil from pulp and paper mils are important sources of sterols, namely sitosterol. The sterol fractions isolated from deodorizates and tall-oil could be eciently used as substrates for the production of steroid precursors through bioconversion with the strain Mycobacterium sp. NRRL B-3805. At present, these wastes have little economic value in Portugal. Deodorizates are usually discharged jointly with other wastes from oil processing, forming an euent with a very high organic load contributing to water pollution problems. In some cases, they are incorporated in animal feed. Tall-oil is burned for energy at Portucel production sites. However tall-oil burning produces corrosive ashes, and adds to air pollutant emissions. A pro®table application of these wastes could be the production of sterol-rich fractions, possibly among other valuable lipidic concentrates, reducing the pollution loads. On the basis of the amount of wastes produced annually in Portugal by the pulp and paper industries, it can be estimated, in case of full tall-oil utilization, that it would be possible to obtain over 90 tons of sterol concentrate per year, with the characteristics given in Table 3, employing the described process P2. References Ahmad, S., Garg, S.K., Johri, B.N., 1992. Biotransformation of sterols: selective cleavage of the side chain. Biotechnol. Adv. 10, 1±67. Angelova, B., Schmauder, H.-P., 1999. Lipophilic compounds in biotechnology ± interaction with cells and technological problems. J. Biotechnol. 67, 13±32. Blunden, G., Culling, M.C., Jewers, K., 1975. Steroidal sapogenins: a review of actual and potential plant sources. Trop. Sci. 17 (3), 139± 154. Castang, J., Olle, M., Derbesy, M., Estienne, J., 1976. Composition de la fraction sterolique de quelques huiles alimentaires. Ann. Fals. Exp. Chim. 69 (737), 57±85. Coelho, J.A.P., Mendes, R., Dias, A.C.P., Cabral, J.M.S., Palavra, A.M.F., Novais, J.M., 1992. Extraction of natural products with supercritical CO2 . In: Balny, C., Hayashi, R., Heremans, K., Mason, P. (Eds.), High Pressure and Biotechnology, vol. 224. Colloque INSERM/John Libbey & Co, London, pp. 439±441. Conner, A.H., Rowe, J.M., 1975. Neutrals in southern pine tall oil. JAOCS 52, 334±338. Dias, A.C.P., Barroso, J., 1990. Utilizacß~ao de resõduos industriais como fonte de esterois para a ind ustria farmac^eutica. In: Proceed-
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ings of V Congresso Nacional de Biotecnologia. Braga, Portugal, PS11. Gy ure, I., Lenkey, B., Szentirmai, A., 1993. Propyonil-CoA elimination may be a rate-determining step of selective cleavage of sterol side chain. Biotechnol. Lett. 15 (9), 925±930. Harvala, T., Alkio, M., Komppa, V., 1987. Extraction of tall oil with supercritical carbon dioxide. Chem. Eng. Res. Des. 65, 386± 389. Heltzel, C.E., Gunatilaka, A.A.L., Kingston, D.G.I., 1994. Synthesis and structure-activity relationships of cytotoxic 7-hydroxy sterols. J. Nat. Prod. 57 (5), 620±628. Issidorides, C.H., Kitagowa, I., Mosttig, E., 1962. Cleavage of steroidal digitonides in dimethyl sulfoxide. J. Org. Chem. 27, 4693±4694. Ivanov, S., Zlatanov, M., 1980. Method for complex processing of the unsaponi®able part of distillation residues from soapstockderived fatty acids and tall-oil. Maslo-Sapunema Prom-st 16 (2), 10±25. Johansson, A.A., Kivikari, R.H.K., Suokas, E.U., 1977. Process for the sterol separation of sterols. US Patent 4,044,031. Karpus, G., Nekrasova, V.B., Kovalev, V.E., 1976. Comparison of LFEA (Leningrad Forrestry Engineering Academy) methods for obtaining sterol products from sulfate soap and tall pitch. Izv. Vyssh. Uchebn. Zaved. Lesn. Zh. 19 (1), 115±118. Kassebi, A., Rousseau, R.W., Kilpatrick, P.K., 1987. Tall-oil recovery and the eects of solid contents, b-sitosterol, acetic acid, and licor source. TAPPI J. (March), 123±125. Kieslich, K., 1985. Microbial side-chain degradation of sterols. J. Basic Microbiol. 25, 461±474. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265±275. Mahato, S.B., Garai, S., 1997. Advances in microbial steroid biotransformation. Steroids 62, 332±345. Marsheck, W.J., Kraychy, S., Muir, R.D., 1972. Microbial degradation of sterols. Appl. Microbiol. 23 (1), 72±77. Maxon, W.D., 1985. Steroid bioconversions: one industrial perspective. In: Tsao, G.T., Flickinger, M.C., Fin, R.K. (Eds.), Annual Reports on Fermentation Processes, vol. 8. Academic Press, New York, pp. 170±185. Minorska, A., Mazgajska, I., 1988. Industrial waste materials as a source of sterols. Fat Sci. Technol. 90 (6), 231±233. Nagasawa, M., Watanabe, N., Hashiba, H., Tamura, G., Arima, K., 1970. Microbial transformation of sterols. Agri. Biol. Chem. 34 (5), 798±800. Neef, G., Eder, U., Seeger, A., Wiechert, R., 1980. Aufbau von Pregnanseitenketten. Chem. Ber. 113, 1184±1188. Nekrasova, V.B., Lapina, E.I., Mednikova, F.A., Agranat, A.L., 1973. Phytosterol obtained from the tall oil made at the Kotlas pulp and paper combine. Izv. Vyssh. Ucheb. Zavec. Les. Zh. 16 (1), 130±133. Perez, C., Perez, I., Herve, E., 1995. Isolation and partial characterization of a new mutant for sterol biotransformation in Mycobacterium sp. Biotechnol. Lett. 17 (11), 1241±1246. Schmid, A., Dordick, J.S., Hauer, B., Kiener, A., Wubbolts, M., Witholt, B., 2001. Industrial biocatalysis today and tomorrow. Nature 409, 258±268. Sedlaczek, L., 1988. Biotransformations of steroids. Crit. Rev. Biotechnol. 7 (3), 187±236. Seidel, L., H orhold, C., 1992. Selection and characterization of new microorganisms for the manafacture of 9-OH-AD from sterols. J. Basic Microbiol. 32 (1), 49±55. Srivastava, S.K., Srivstava, R.A.K., Mathur, S.N.., 1985. Biotransformation of sugarcane sterols into androsta-1,4-diene-3,17-dione (ADD) by Arthrobacter globiformis str. oxydans. J. Appl. Bacteriol. 59, 399±402. Szentirmai, A., 1990. Microbial physiology of sidechain degradation of sterols. J. Ind. Microb. 6, 101±116.
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Szykula, J., Hebda, C., Orpiszewski, J., Saganska, K., 1991. Microbial transformation of neutral fraction and upgraded neutral fraction of Polish tall-oil. Biotechnol. Lett. 13 (12), 917±922. Touche, J., Derbesy, M., Cas, M., Estienne, J., 1975. Dosage des sterols dans les corps gras. Ann. Fals. Exp. Chim. 68 (726), 101±112.
Van Rheenen, V., Shephard, K., 1979. New synthesis of corticosteroids from 17-keto steroids: application and stereochemical study of the unsaturated sulfoxide±sulfonate rearrangement. J. Org. Chem. 44, 1582±1584.
Isolation of a biodegradable sterol-rich fraction from industrial wastes A.C.P. Dias a, P. Fernandes
b,c
, J.M.S. Cabral b, H.M. Pinheiro
b,*
a Departamento de Biologia, Universidade do Minho, Campus de Gualtar, Braga 4710-057, Portugal Centro de Engenharia Biol ogica e Quõmica, Instituto Superior T ecnico, Av. Rovisco Pais, Lisboa 1049-001, Portugal Departamento de Engenharias e Tecnologias, Universidade Lus ofona de Humanidades e Tecnologias, Av do Campo Grande, Lisboa 1749-024, Portugal b
c
Received 15 September 2001; accepted 8 October 2001
Abstract Several industrial waste materials were screened for their sterol content. The possibility of using these industrial by-products as sterol sources for the microbiological production of 4-androsten-3,17-dione (AD) and 1,4-androsta-diene-3,17-dione (ADD) was investigated. Two methods of obtaining the sterol fraction from wastes were developed. Sterol-rich (96±98%) fractions were isolated in a yield above 70%, from a tall-oil euent of a paper pulp industry and from edible-oil deodorizates. These fractions were subsequently used as a substrate for microbial degradation by a Mycobacterium sp. strain and proved to be easily converted to AD and ADD. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Tall-oil; Sitosterol; Oil deodorizates; Mycobacterium sp.; Biotransformation; 4-Androstene-3,17-dione
1. Introduction Although steroid drugs represent only a small part of the world market of pharmaceuticals, there is a great demand for new and cheaper steroid raw materials for their production (Kieslich, 1985; Sedlaczek, 1988; Ahmad et al., 1992; Mahato and Garai, 1997; Angelova and Schmauder, 1999; Schmid et al., 2001). The choice of starting material has always had a critical impact on the steroid-manufacturing industries. The most common and economical process for the production of steroid pharmaceuticals is the partial synthesis from relatively inexpensive steroid raw materials of animal and plant origins. Diosgenin and other sapogenins (e.g., hecogenin) were the preferred starting materials up to the 1970s. However, the protection of Dioscorea plants reduced the availability of diosgenin and increased its price. Soybean sterols obtained from soybean oil processing were plentiful and cheap and included a large fraction of stigmasterol which can be easily converted to progesterone, thus being an excellent alternative to diosgenin (Maxon, 1985). Blunden et al. (1975) estimated that stigmasterol *
Corresponding author. Tel.: +351-1-841-9125; fax: +351-1-8419062. E-mail address: [email protected] (H.M. Pinheiro).
represented about 15% of the total precursors used in the USA, becoming, with diosgenin, one of the main raw materials for industries producing steroids of the pregnane, androstane and estrane series. Whereas stigmasterol could be degraded chemically starting with the oxidative cleavage of the 24-double bond, preserving the steroid ring structure, sterols like sitosterol and cholesterol, with saturated side-chains resistant to selective degradation, were considered low-valued or even waste products. However, since the isolation of the ®rst mutant Mycobacterium sp. strain capable of degrading the sidechain of sterols giving 17-keto-steroids (Marsheck et al., 1972) and the development of methods for the chemical addition of the corticoid side-chain to these 17-intermediates (Van Rheenen and Shephard, 1979; Neef et al., 1980), b-sitosterol, the most ubiquitous plant sterol, became a major raw material for the synthesis of corticosteroids, which represent the gross of the steroid industry (Sedlaczek, 1988). As an alternative to puri®ed sitosterol, mixed sterol concentrates obtained from natural sources such as soya (Marsheck et al., 1972) or rape seed (Schmid et al., 2001) or from industrial wastes (sugarcane and paper industries) (Srivastava et al., 1985; Perez et al., 1995; Szykula et al., 1991) have been tested as substrates for the microbial production of 17-ketosteroids. Over 1000 tons of the latter chemicals are
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 1 ) 0 0 1 8 7 - 0
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produced per year (Schmid et al., 2001). Additionally, sitosterol can be converted by other microbial mutants to 9a-hydroxy-17-keto-steroids (Sedlaczek, 1988; Seidel and H orhold, 1992) or 20-carboxy pregnane derivatives (Szentirmai, 1990) which are more suitable for corticosteroid synthesis. This paper reports on a screening of several wastes from Portuguese industries as potential sources of sterol mixtures, rich in b-sitosterol, for microbial conversion to 4-androstene-3,17-dione (AD), a convenient raw material for the steroid industry. The isolation of the sterol fraction from these wastes and the use of these sterol fractions as substrates for microbial side-chain cleavage were investigated.
2. Methods 2.1. Waste samples Tall-oil and sulfate pulp samples were a gift from Portucel ± Empresa de Celulose e Papel de Portugal SGPS, SA, from factories located at Viana do Castelo and Vila Velha do Rod~ ao, Portugal. Black liquor samples were supplied by Celulose do Caima SA (Const^ ancia, Portugal). Deodorizates from edible-oil re®ning processes were supplied by several Portuguese companies, with varied plant origins: soybean (Iberol ± Sociedade Iberica de Oleaginosas, SA, Alhandra, Portugal), sun ¯ower (Tagol ± Companhia de Oleaginosas do Tejo, SA, Almada, Portugal), peanut (Mendes Godinho, Tomar) and from a mixture of peanut and grape seed (Sovena ± Sociedade Vendedora de Glicerina, SA, Lisboa, Portugal). 2.2. Chemicals Yeast extract and potato dextrose agar (PDA) were from Difco, USA. Sterol and steroid standards were from Sigma, USA. Fructose, glycerol, chloride salts, potassium and sodium hydroxide and phosphate salts were from Merck, Germany. Ethanol, methanol, diethylether, dichloromethane, dimethyl sulfoxide, tetrahydrofurane and sulfuric and hydrochloric acids were from Riedel-de H aen, Germany. Acetone was from Fluka, Switzerland. Ammonium chloride was from J.T. Baker, The Netherlands. Hexane and ethyl acetate were from Labscan, Ireland. All these chemicals were of p.a. grade. 2.3. Production of unsaponi®ables Dried (70 °C, with vacuum, to constant weight) portions of the several waste samples were submitted to saponi®cation with a 13.6 M potassium hydroxide
solution in ethanol (1:8 v/v), in a proportion of 50 g dried waste to 112.5 ml ethanolic solution, under re¯ux, for 2±3 h. The unsaponi®able fraction was extracted three times with one volume of either diethylether or a mixture of n-hexane and ethyl acetate (5:1, v/v). The latter mixture was used when further sterol puri®cation was aimed at. The organic phase was washed with a sodium hydroxide (0.5 M) solution for the removal of acidic residues and then with water to non-alkaline reaction and dried over anhydrous sodium sulfate or calcium chloride. The organic solvent was distilled o at 40±60 °C in a rotary evaporator and the residue was dried to constant weight under vacuum at 70 °C. 2.4. Isolation of sterol-rich fractions Sterol-rich fractions were isolated from the unsaponi®able residue (above) by two distinct methods. In process P1, residue portions (5±200 g) were dissolved in 30±500 ml of petroleum ether, which was then saturated with steam to induce precipitation of the crude sterol fraction. In process P2, the residue portions (20 g) were dissolved in 100 ml of a mixture of hexane, acetone, methanol and water (49.3:34.0:15.1:1.6, v/v). The solution was then concentrated under vacuum at a temperature of 45±50 °C until sterol precipitation. In some trials, and for both processes, following sterol precipitation the remaining solution was evaporated to dryness. A second sterol-rich fraction was obtained from the recovered solid residue by repeating process P1 or P2, respectively. The crude sterol precipitates were recovered by ®ltration on ®lter paper Whatman No. 1, washed with cold (4 °C) n-hexane to remove the bulk of contaminants that precipitated together with the sterols and dried to constant weight under vacuum at 60 °C. 2.5. Biotransformation assays Biotransformations of the sterol-rich fractions isolated were carried out using Mycobacterium sp. NRRL B-3805 in 500 ml Erlenmeyer ¯asks, with a working volume of 100 ml. Bacterial cells were maintained on PDA slants (40 g l 1 ) at room temperature. Prior to fermentation cells were grown in slants for 3 days at 30 °C. The growth and bioconversion medium was composed of (in g l 1 ) fructose or glycerol (10), ammonium chloride (2), KH2 PO4 (0.75), K2 HPO4 (2.11), MgSO4 7H2 O (0.14), CaCl2 (0.025) and sterol-rich fraction (0.4 or 1.0). The inoculum was pre-cultivated in 100 ml Erlenmeyer ¯asks in a liquid medium consisting of yeast extract (10 g l 1 ) in phosphate buer 0.02 M pH 7.0, at 30 °C, with orbital shaking at 200 rpm. When the culture reached an optical density (O.D., 640 nm) of 0.6±0.9 (after 12±17 h), 2 ml were used to inoculate the larger ¯asks. Fermentations were run at 30 °C, with orbital shaking at 150 rpm and pH control at 7:0 0:3
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with 2 M NaOH or HCl solutions. Samples were collected from the liquid medium at several incubation times up to 192 h, extracted three times with ethyl acetate (1:1 v/v) and dried over anhydrous sodium sulfate or calcium chloride. The organic phase was distilled o and sterol/steroid analysis was performed by HPLC, GC, GC±MS. Cell growth was followed by measurement of protein concentration in the aqueous phase of the samples, as described by Lowry et al. (1951), after cell hydrolysis, by heating at 100 °C, for 20 min, in 1 M NaOH (Gy ure et al., 1993). 2.6. Analytical methods The presence of sterols in the samples (unsaponi®able waste raw materials, sterol-rich fractions and dried, hexane-extracted neutral fraction from mother liquors) was semi-quantitatively evaluated, after extraction with or dissolution in diethylether, by thin layer chromatography (TLC). Merck silica gel 60 plates were eluted with a mixture of dichloromethane and methanol (9:1 v/ v) and revealed with concentrated sulfuric acid in ethanol (50:50 v/v) at 100 °C. Sterol content in the unsaponi®able fractions was determined by precipitation of sterol digitonides as described elsewhere (Dias and Barroso, 1990). The sterol digitonides were cleaved with dimethyl sulfoxide as described by Issidorides et al. (1962) and the free sterols recovered. These sterols were dissolved in 200 ll tetrahydrofuran, silylated with 150 ll of hexamethyldisilazane (Merck, Germany) and 50 ll of trimethylchlorosilane (Merck, Germany) at 70 °C for 3 h, and analyzed by GC and GC±MS. Samples of the stero(l)id-rich fraction used for bioconversion were analyzed by HPLC. Quanti®cation was done by the internal standard method using progesterone as standard. Additionally, sterols and steroids were analyzed quantitatively and qualitatively by GC after trimethylsilyl derivatization. Quanti®cation was done by the internal standard method using cholestane as standard. Con®rmation of the identity of the compounds was done by GC±MS. Sterol consumption and steroid production during bioconversion were evaluated by HPLC and GC analysis. Samples were taken from the bioconversion media and extracted with two volumes of ethyl acetate. The organic phase was recovered and the solvent evaporated. The remaining stero(l)id residue was ®nally dissolved in a solution of progesterone (0:2 g l 1 ) in n-heptane (HPLC analysis) or dissolved in a solution of cholestane (0:1 g l 1 ) and silylated as described above (GC analysis). HPLC analyses were performed in a Lichrosorb Si-60 column (250 mm 40 mm; 10 lm particle diameter), with n-heptane containing 6% (v/v) ethanol as the mobile phase, at a ¯ow rate of 1:0 ml min 1 . Total sterol was detected at 215 nm and steroid products at 254 nm,
255
and matched to pure standards. For total sterol, the standard was the adequate sterol-rich fraction, previously identi®ed and quanti®ed by GC±MS. GC analyses were done in a Perkin±Elmer 8600 gas chromatograph equipped with an FID detector, a datahandling system and a DB-1 fused-silica column (30 m 0:25 mm i.d., ®lm thickness 0.25 lm). The oven temperature was programmed in a ®rst slope from 200 to 220 °C, at 2 °C min 1 , and in a second from 220 to 280 °C at 5 °C min 1 , ®nally remaining at 280 °C for 30 min. Injector and detector temperatures were 300 and 320 °C, respectively. The carrier gas was H2 adjusted to a linear velocity of 300 cm s 1 . Samples were injected using the split-sampling technique with a ratio of 1:150. For GC±MS analyses the chromatograph was equipped with a DB-1 fused-silica column (30 m 0:25 mm i.d., ®lm thickness 0.25 lm) and interfaced with an iontrap detector (Finnigan Mat). Oven conditions were as described above. Transfer line and ion-trap temperatures were 280 and 220 °C, respectively. The carrier gas was He adjusted to a linear velocity of 30 cm s 1 . The split ratio was 1:40 and the ionization voltage was 70 eV, the scan range 40±500 mu and the scan time 1 s. Mass spectra and the reconstructed chromatogram were obtained by automatically scanning in the mass range m=z from 20 to 600 a.m.u. Retention time and mass spectra of the compounds in the samples were compared with those of authentic standards for identi®cation. 3. Results and discussion 3.1. Characterization of wastes in terms of sterol quality and content Earlier studies reported that tall-oil obtained as a byproduct in kraft pulp and paper mills (Conner and Rowe, 1975; Minorska and Mazgajska, 1988) as well as the wastes from the deodorization of vegetable oils (Minorska and Mazgajska, 1988) can be convenient sources of sterols, namely b-sitosterol. Therefore, several wastes from Portuguese paper pulp and edible-oil companies were examined in order to evaluate their potential importance as sterol raw material sources for the pharmaceutical industry. The results of the primary characterization of the sampled wastes are shown in Table 1. TLC analysis of all these wastes, except the black liquor from a eucalyptus pulp mill, revealed substantial amounts of sterols. The unsaponi®able matter found in Portucel tall-oil (12.7% w/w) was in agreement with the typical unsaponi®able percentages (10±40%) found in tall-oil from hardwoods (Kassebi et al., 1987). Portucel tall-oil sterol content (4.3% w/w) was similar to those found in the literature (Nekrasova et al., 1973; Conner and Rowe, 1975) and almost 10 times higher than that of Polish
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Table 1 Characteristics of examined waste samples Raw material
Water (%)
Unsaponi®able matter (%)
Sterol in unsaponi®able matter (%)
Sterol in raw material (%)
Pulp and paper Sulfate soap Tall-oil Black liquor
29 1 ngl 96 1
7.6 0.2 12.7 0.3 0.3 0.1
25 1 34 1 nd
1:4 0:2 4:3 0:2 nd
Edible-oil deodorizates Peanut/grape seed Peanut Sun¯ower Soybean
ngl ngl ngl ngl
21.1 0.2 18.0 0.3 14.3 0.2 27.1 0.3
13 2 18 1 22 0:5 20 1
2:9 0:3 3:3 0:1 3:2 0:1 5:4 0:2
All the () values were calculated on a dry weight basis and correspond to an average of at least four determinations. Sterol content was determined by the digitonine method (see materials and methods); ngl ± negligible values; nd ± sterols not detected by TLC.
tall-oil (Minorska and Mazgajska, 1988). The sterol content of tall-oil, on a dry weight basis, is nearly triple that found in sulfate soap, which is in agreement with the conversion yield obtained by Portucel (1 ton of talloil obtained from 3 tons of sulfate soap). Oil deodorizates have larger amounts of unsaponi®able matter than pulp wastes, varying from 14% to 27% (Table 1). Soy edible-oil deodorizates from Iberol also present the highest sterol content among the studied wastes. Both the unsaponi®able and sterol amounts present in deodorizate wastes are signi®cantly larger than those found in Polish deodorizates (Minorska and Mazgajska, 1988). This could be related either to dierences in the deodorization process or to the origin of the starting plant material, admitting negligible analytical discrepancies, since the methodology followed for sterol quanti®cation was very similar to the one used by the Polish authors. The unsaponi®able fractions obtained were analyzed by GC±MS and the majority of the sterols present were identi®ed (Table 2). Campesterol and b-sitosterol were present in all wastes, the latter as the main sterol present, irrespective of the waste origin. However, a signi®cant dierence in sterol composition was
observed when pulp and paper wastes or oil deodorizates were analyzed. b-Sitostanol was only detected in pulp and paper wastes, whereas stigmasterol was present in signi®cant amounts only in oil deodorizates. Sterol compositions of Portucel pulp wastes were very similar, as expected, since tall-oil is obtained from sulfate soap, and was composed mostly of b-sitosterol, in accordance with the reported sterol composition of other tall-oils (Nekrasova et al., 1973; Conner and Rowe, 1975; Szykula et al., 1991) and to that found in Portuguese Pinus pinaster whole plants which are the main wood raw materials used (results not shown). Furthermore, the processing of sulfate soap only concentrates the sterol fraction, thus not changing the relative sterol amounts. Deodorizates have sterol fractions with less b-sitosterol (46±69%) particularly the soy deodorizate (Iberol), and with much more campesterol and stigmasterol (Table 2). The presence of a relatively high content of campesterol and stigmasterol in soy deodorizates is in agreement with sterol soy oil composition which has, comparatively to peanut and sun¯ower oils, higher amounts of stigmasterol and campesterol and lower b-sitosterol amounts (Touche et al., 1975; Castang et al., 1976).
Table 2 Sterol composition of the unsaponi®able fractions of the raw materials examined Raw material
b-Sitosterol
Pulp and paper Sulfate soap Tall-oil
78 78
Oil deodorizates Peanut/grape seed Peanut Sun¯ower Soybean
62 66 69 46
Campesterol 6 5.5 12 13 13 27
Stigmasterol
b-Sitostanol
tr tr
11 11.5
12 13 14 24
nd nd nd nd
Other sterolsa 5 5 14 8 4 3
Values are expressed as the percentage by weight on the basis of the total free sterols obtained by the cleavage of the unsaponi®able fraction digitonides. The values represent the average of three independent replicates (tr ± trace amounts; nd ± not detected). a As determined by GC±MS and compared to library spectra.
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3.2. Isolation of sterol-rich fractions from wastes The use of these wastes as a source of sterols for the steroid industry required further processing of the unsaponi®able fractions in order to obtain sterol-rich fractions allowing the development of eective processes for the microbial biotransformation to corticosteroid precursors such as 4-androsten-3,17-dione (AD) and 1,4-androsten-3,17-dione (ADD). The microbial degradation of an unsaponi®able crude fraction from tall-oil led to lower bioconversion yields in AD and ADD and to a rather complex puri®cation process for the recovery of the products, as compared to the use of an upgraded substrate fraction (Szykula et al., 1991). The production of these sterol-rich fractions was achieved for deodorizates as well as for tall-oil wastes using the processes P1 and P2 described above. The results summarized in Table 3 show that in only one step, very pure sterol fractions (sterol content above 96%) could be obtained from unsaponi®able matter with high yield (above 70%). The fractions are composed of white to pale-yellow crystals with sterol compositions similar to those found in the starting unsaponi®able fractions. However some sterol remained on the mother liquors as revealed by TLC. A second precipitation of the mother liquors was carried out, using both methods, leading to second sterol-rich fractions, increasing the total sterol recovery yield up to 93% (results not shown). However these fractions were less pure, with a sterol content in the 85± 90% range and a yellow brown color. On the other hand, the use of the P1 process with tall-oil unsaponi®able fractions was found inappropriate since it gave a low purity sterol fraction in also low yield, showing that the P2 process is preferable for this kind of waste. The P2 process is a modi®cation of the protocol outlined by Johansson et al. (1977). In this process the unsaponi®able matter was ®rst dissolved in a mixture of hexane, acetone and methanol and submitted to liquid±liquid extraction of the polar compounds, including the sterols, into a hydrophilic solvent mixture, prior to crystallization of the sterol fraction, giving similar yields to those obtained in this work. Other methods referred the production of sterol-rich fractions, with a lower sterol content relatively to those obtained in the present case,
257
by complexing the sterols in sulfate soap and unsaponi®able fractions from tall-oil and oil deodorizates with urea and zinc chloride, respectively (Karpus et al., 1976; Ivanov and Zlatanov, 1980). Direct supercritical carbon dioxide extraction of tall-oil seems to be selective enough to separate the neutrals, rosins and fatty acids present in tall-oil, thus isolation and puri®cation of more valuable components, e.g., b-sitosterol and squalene from neutrals, may be possible (Harvala et al., 1987). Supercritical extraction of the Portucel tall-oil unsaponi®able fraction was attempted, aimed at obtaining a sitosterol-rich fraction. Although an increased sitosterol concentration was measured in the extract, the concentration values were much lower than those obtained with the P2 process (Coelho et al., 1992). Nevertheless a previous increase in sterol concentration through supercritical extraction could help in the subsequent production of a sterol-rich fraction by the P2 process. 3.3. Bioconversion of the sterol-rich fractions One of the possible interesting uses of the sterol-rich fractions obtained is the microbial side-chain degradation to 17-keto-compounds like AD or ADD. AD and ADD are currently used as starting materials for the synthesis of corticosteroids and 19-nor-steroids, respectively (Kieslich, 1985; Ahmad et al., 1992). Sterol-rich fractions isolated from Polish tall-oil with a sterol content of approximately 20% and 84% were successfully converted by Mycobacterium sp. MB-3683 (Szykula et al., 1991). The sterol-rich fractions isolated from tall-oil unsaponi®ables and sun¯ower oil deodorizates were used in biotransformation runs with Mycobacterium sp. NRRL B-3805, a microorganism that degrades sterols to AD (the major product) and ADD (Marsheck et al., 1972). The microbial strain eectively cleaved the side-chain of the isolated sterols to AD and ADD, particularly when talloil unsaponi®able isolates were used (Table 4). The lower product yield obtained with oil deodorizate isolates could be related to their relative sterol composition. Large amounts of stigmasterol, which has a D22 -double bond in the side-chain, can be found in these deodorizates. This
Table 3 Yield and sterol content of the sterol-rich fractions obtained from the unsaponi®able fraction of tall-oil and oil deodorizate wastes, by the isolation processes described in materials and methods
a
Raw material
Isolation process
Sterol-rich fraction recovery yielda (%)
Sterol contentb (%)
Tall-oil Sun¯ower Soybean
P2 P1 P1
72 2 75 2 72 2
>96 >96 >96
Values on the basis of the total sterol amounts present in the unsaponi®able fractions (determined by the digitonine method). The values represent the minor and major values obtained from four independent replicates. b Sterol content of the isolated sterol-rich fraction on a dry weight basis (GC method), obtained in three independent isolation experiments.
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Table 4 Bioconversion and product yields of the microbial degradation of sterol-rich fractions isolated from tall-oil and sun¯ower oil processing wastes Sterol-rich fraction
Sterol-rich fraction concentration (mM)
Carbon source
Final conversion yielda (%)
Final product yieldb (%)
Sun¯ower Tall-oil Tall-oil Tall-oil
1.0 1.0 2.5 2.5
Fructose Fructose Glycerol Fructose
52 5 65 4 52 5 42 1
41 1 55 2 45 2 16 1
Values are averages from at least two independent bioconversion runs. a Calculated from sterol consumption on a molar basis. b Calculated from AD+ADD production from the initial sterol, on a molar basis.
double bond renders microbial degradation more dicult in a general way (Nagasawa et al., 1970) and the conversion of stigmasterol to androstanes by Mycobacterium sp. NRRL B-3805 has been reported to be lower than that observed with sitosterol (Marsheck et al., 1972). Indeed we observed a lower degree of conversion for stigmasterol than that observed for b-sitosterol (Fig. 1). Therefore, tall-oil wastes are the best choice for obtaining sterol-rich fractions for further bioconversions since all the main sterols present in the isolates are easily transformed. In order to enhance the process productivity, initial substrate concentrations were increased from 1.0 to 2.5 mM. Similar product yields could be obtained, as long as glycerol replaced fructose as carbon source. Possibly dierent carbon sources induce dierent metabolic activities or lead to dierent cell envelope characteristics, interfering with substrate mass transfer. Further studies are required for process optimization. Figs. 2 and 3 show a typical time course of the degradation of the sterol fractions isolated from tall-oil and sun¯ower oil deodorizates. The results in Fig. 2 con®rm that the main sterols that compose these fractions, respectively, b-sitosterol, b-sitostanol and campesterol, were metabolizable and led to AD/ADD accumulation, as veri®ed by HPLC and GC±MS analysis.
Fig. 2. Bioconversion of the sterol-rich fraction from the unsaponi®ables of tall-oil, using Mycobacterium sp. NRRL B-3805, with fructose as carbon source. b-Sitosterol (N), campesterol (d), b-sitostanol (j), AD ( ) and ADD () concentrations are shown.
Fig. 3. Bioconversion of the sterol-rich fraction from sun¯ower deodorizates, using Mycobacterium sp. NRRL B-3805, with fructose as carbon source. Overall sterol concentration (j), AD ( ) and ADD () concentrations are shown.
Fig. 1. Bioconversion of the sterol-rich fraction from the unsaponi®ables of tall-oil, using Mycobacterium sp. NRRL B-3805, with glycerol as carbon source. b-Sitosterol (N), campesterol (d), b-sitostanol (j), AD ( ), and ADD () concentrations are shown.
In order to improve the bioconversion yields of sterol-rich fractions isolated from oil deodorizates, particularly soy oil, a liquid±liquid extraction step to recover stigmasterol could be introduced. The stigmasterol thus obtained could be converted by other processes to obtain precursors for the steroid industry (Maxon, 1985) or for the synthesis of cytotoxic 7-hy-
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droxy sterols (Heltzel et al., 1994). Alternatively, the use of strains such as Mycobacterium EX-4, a mutant from Mycobacterium sp. B-3683 showing a high ability for the bioconversion of stigmasterol (Perez et al., 1995), could be preferred for the biodegradation of sterol-rich fractions from oil deodorizates.
4. Conclusion The analysis of the dierent industrial wastes considered in this study has shown that deodorizates from edible-oil re®ning and sulfate soap or tall-oil from pulp and paper mils are important sources of sterols, namely sitosterol. The sterol fractions isolated from deodorizates and tall-oil could be eciently used as substrates for the production of steroid precursors through bioconversion with the strain Mycobacterium sp. NRRL B-3805. At present, these wastes have little economic value in Portugal. Deodorizates are usually discharged jointly with other wastes from oil processing, forming an euent with a very high organic load contributing to water pollution problems. In some cases, they are incorporated in animal feed. Tall-oil is burned for energy at Portucel production sites. However tall-oil burning produces corrosive ashes, and adds to air pollutant emissions. A pro®table application of these wastes could be the production of sterol-rich fractions, possibly among other valuable lipidic concentrates, reducing the pollution loads. On the basis of the amount of wastes produced annually in Portugal by the pulp and paper industries, it can be estimated, in case of full tall-oil utilization, that it would be possible to obtain over 90 tons of sterol concentrate per year, with the characteristics given in Table 3, employing the described process P2. References Ahmad, S., Garg, S.K., Johri, B.N., 1992. Biotransformation of sterols: selective cleavage of the side chain. Biotechnol. Adv. 10, 1±67. Angelova, B., Schmauder, H.-P., 1999. Lipophilic compounds in biotechnology ± interaction with cells and technological problems. J. Biotechnol. 67, 13±32. Blunden, G., Culling, M.C., Jewers, K., 1975. Steroidal sapogenins: a review of actual and potential plant sources. Trop. Sci. 17 (3), 139± 154. Castang, J., Olle, M., Derbesy, M., Estienne, J., 1976. Composition de la fraction sterolique de quelques huiles alimentaires. Ann. Fals. Exp. Chim. 69 (737), 57±85. Coelho, J.A.P., Mendes, R., Dias, A.C.P., Cabral, J.M.S., Palavra, A.M.F., Novais, J.M., 1992. Extraction of natural products with supercritical CO2 . In: Balny, C., Hayashi, R., Heremans, K., Mason, P. (Eds.), High Pressure and Biotechnology, vol. 224. Colloque INSERM/John Libbey & Co, London, pp. 439±441. Conner, A.H., Rowe, J.M., 1975. Neutrals in southern pine tall oil. JAOCS 52, 334±338. Dias, A.C.P., Barroso, J., 1990. Utilizacß~ao de resõduos industriais como fonte de esterois para a ind ustria farmac^eutica. In: Proceed-
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