Abstract: A total of 75 different isolates of filamentous fungi were screened for their respective ability to accumulate lovastatin. Twenty, sixteen and seven isolates ...
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Bioproduction of lovastatin (mevacor; a hypocholesterolemic drug) by some fungi using agro-industrial by-products A. A.Zohri, I. A. El-Kady, and Shimaa Ragab Botany Department, Faculty of Science, Assiut University, Assiut, Egypt.
Abstract: A total of 75 different isolates of filamentous fungi were screened for their respective ability to accumulate lovastatin. Twenty, sixteen and seven isolates proved to be low, moderate and high lovastatin producers. Generally high lovastatin levels were obtained using shaking cultivation than those recorded when static cultivation was used. The optimal nutritional conditions for maximum production (520 mg/l) of lovastatin by P. camemberti No. 157 (the superior lovastatin producer) were: 150 g/l glucose and 2.5 g/l ammonium nitrate as carbon and nitrogen sources, respectively, while the optimal pH value, temperature and incubation period were pH 5.5, 30oC and 12 days, respectively. A trail for the utilization of 15 agro-industrial wastes or byproducts for lovastatin production by the five selected highly producer isolates was made. The isolates have the ability to grow and produce lovastatin, with a range fluctuated between 150-410, 160-370 and 155-280 mg/l on pea, kidney bean seeds and mixed vegetables wastes, respectively. The highest level of lovastatin formed on pea waste was obtained by P. camemberti No. 157. Four fungal isolates only could grow and produce lovastatin at concentrations ranged between 140 and 410 mg/l on carrot waste while the five fungal isolates under test grew on turnip waste and produced relatively low levels of lovastatin. Levels of lovastatin produced by the five fungal isolates ranged from 210-430, 200-380, 156-280 and 260-520 mg/l on apple, apricot, orange and peach wastes, respectively. The highest levels of lovastatin recorded on each of apple (430 mg/l) and peach (520 mg/l) by P. camemberti No. 157 while those recorded on each of apricot (380 mg/l) and orange wastes (280 mg/l) formed by P. multicolor No. 221. Lovastatin concentrations produced on the three agriculture by-products media (wheat bran, rice fragment and rice husk) were relatively high and fluctuated between 190-510 mg/l on wheat bran, 180-520 mg/l on rice fragment and 220-520 mg/l of rice husk media. The highest amounts of this drug were produced by P. camemberti No. 157 on each of the three kinds of agricultural by-products. The five fungal isolates tested grow well and produce relatively moderate lovastatin levels (from 145 to 360 mg/l) when molasses used as nutritive medium. Low levels of lovastatin were formed on each of corn steep liquor and cheese whey media by each of P. spinulosum No. 232, F. moniliforme No. 276. Penicillium camemberti No. 157 produced relatively high levels (120-310 mg/l of this compound on corn steep liquor, molasses and cheese whey media. An attempt for production of lovastatin on semi-industrial scale using a 1.5 liter laboratory fermentor has been made. P. camemberti No. 157 on rice fragment medium was made and
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grown. The maximum level (620 mg/l) of lovastatin was obtained at the ninth and tenth days of fermentation.
KEY WORD: Lovastatin, Mevacor, Hypocholestemic drug, Bioproduction, Fungi, Agro-industrial by-products. Introduction: Hypercholesterolemic is the accumulation of cholesterol in blood plasma that causes atherosclerosis (blockage of the artery), leading to the coronary heart disease and heart attack (Endo, 2004; Valera et al., 2005). Out of total body cholesterol, one-third comes from diet and nearly twothird is synthesized in the body. So, inhibition of de novo synthesis would be expected to lower plasma cholesterol. Lovastatin (= mevinolin = mevacor) was the first hypocholesterolemic drug to be approved by United States Food and Drug Administration (FDA) (Manzoni and Rollini, 2002). It competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG. CoA) reductase, the rat-limiting enzyme of cholesterol biosynthesis and lower plasma cholesterol in human and animals (Alberts, 1988). Recently, lovastatin has also been reported as a potential therapeutic agent for the treatment of various types of tumor because it suppresses tumor growth in vivo by inhibiting the synthesis of non-sterol isoprenoid compounds (Jones et al., 1994; Newman et al., 1994). A screen of 8,000 strains of microorganisms for their ability to produce an inhibitor of in vitro sterol synthesis by rat liver enzyme system resulted in the isolation of mevastatin (Endo, 1985). A search for other compounds which also inhibited cholesterol synthesis resulted in the isolation of lovastatin. Lovastatin is produced by A. terreus (ATCC 20542) as recorded by Alberts et al. (1980). It is also produced by Monoascus rubber (Endo, 1979). A fermentation process with A. terreus was developed to manufacture it on large scale (Buckland et al., 1989). Utilization of some agro-industrial wastes or by-products for the microbial production of useful products has been recommended by many investigators in our laboratory such as: using sugar cane molasses for ergosterol by filamentous fungi (El-kady et al., 1994; Eman Mostafa and Zohri, 1997), whey for glycerol production by different members of fungi (Zohri, 2000) sugar can molasses for steroid transformation by fungi (Abdel-Galil, 2000) and using wastes of orange, potatos, peach, wheat steep liquor and molasses for cyclosporin A production by selective filamentous fungi (Rgaa Kotby, 2006). Thus the objective of this study was designed to study: the potentialities of lovastatin production by 75 different fungal isolates of filamentous fungi; comparison between static and shaking cultivation methods for lovastatin productions; optimization of both nutritional and 4-7 March 2007, Sharm El Sheikh, Egypt
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environmental factors affecting lovastatin formation; utilization of 15 kinds of agro-industrial wastes or by-products for formation of lovastatin as well as production of this drug on semi-industrial scale using a laboratory fermentor. MATERIALS AND METHODS: Tested Fungal Isolates: Seventy-five isolates of thirty-seven species and two species varieties represented ten genera of filamentous fungi were examined in this study. These isolates were obtained from Botany Department, Faculty of Science, Assiut University, Egypt and AUMC (Assiut University Mycological Center) Assiut University, Egypt. Medium and Fermentation: Each isolate was cultivated on modified liquid Czapek’s medium of the following composition (g/l of distilled water): glucose, 100.0; sodium nitrate, 2.0; potassium dihydrogen phosphate, 1.0; magnesium sulphate, 0.1 and supplemented with yeast extract, 3.0; malt extract, 3.0 and peptone. 3.0 g/l (Safaa Mahmoud, 2001). The cultures were grown in 250 ml Erlenmeyer flasks, each containing 50 ml of the synthetic medium. The flasks were sterilized at 121 oC for 20 min and inoculated after cooling with 2 ml spores suspension of 7-10 days old cultures. The cultures were incubated at 28oC as stationary cultivation for 10 days. Agro-industrial Wastes or by-Products Used as Culture Media: a) Fruits, vegetables & pickles wastes and three agriculture byproducts : Each fungal isolate of the highly lovastatin producers (seven isolates) was cultivated on different semi-synthetic culture media. Each medium contained 100.0 g of agro-industrial waste or by-product, individually, of each of orange, peach, apple and apricot as fruit wastes; pea, mixed vegetable and kidney beans as vegetables wastes; carrot, and turnip as pickle wastes as well as wheat bran, rice fragments and rice husk as agriculture byproducts added to one liter of distilled water and supplemented with 2.5 g/l of ammonium nitrate. All industrial wastes were collected from different juices, vegetables canning and pickles factories located at the industrial areas of different governorates in Egypt, while the three agriculture byproducts were collected from farms of different governorates in Egypt. b) Corn steeps liquor: Corn steeps liquor was prepared by using sweet corn maize. 100 g from the substrate was put in 2000 ml Erlenmeyer flasks and completed to 2000 ml by distilled water and cooked on a very quiet flame for 12 h, after 4-7 March 2007, Sharm El Sheikh, Egypt
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that, these were filtered through a muslin cloth and used. Each individual fungal isolate of the highly lovastatin producers was cultivated on a medium consist of 100.0 ml of corn steep liquor added to 900 ml of distilled water to complete one liter medium. c) Cheese whey: Salted cheese whey is a by-product formed from milk during the production of cheese (both soft and hard cheese). Whey used during this work was produced from milk composed of 1:1 cow’s and buffalo’s milk which used for production of white soft (Domiati type) cheese. Whey sample was kindly provided by Dairy Department, Faculty of Agricultural, Assiut University. Samples of whey (8% NaCl) were centrifuged (5000 rpm, 10 min), the sediment was discarded, and samples of supernatant were used as it is. d) Black-strap molasses: Black-strap molasses supplemented from El-Hawamdya sugar cane factory were tested as natural medium for cultivation of the experimental organisms. The molasses sample was centrifuged (5000 rpm, 10 min), the muddy sediment was discarded, and samples of supernatant were tested. Each individual fungal isolate of the highly lovastatin producers was cultivated on liquid semi synthetic medium of the following composition: molasses, 100 ml; ammonium nitrate, 2.5 g and completed to one liter distilled water. The pH of the different media was adjusted at 5.5 before sterilization. The cultures were incubated at 28±2oC on rotary shaker (220 rpm) for 12 days. Lovastatin Extraction: At the end of fermentation period, the content of each flask (medium and mycelium) were homogenized in a blender (16000 rpm), the mixture was adjusted to pH 3 with 2N HCl and rehomogenized with double its volume of ethyl acetate (100 ml) as described by Serizawa et al. (1983). The extraction was repeated three times in order to ensure that all the products were extracted. The combined ethyl acetate were washed with half its volume of 5% sodium bicarbonate solution; followed by an equal volume of distilled water, dried over anhydrous sodium sulphate, filtered, then distilled to give a semisolid residue (test material). Lovastatin Determenation: Lovastatin was identified using thin layer chromatography (Stahl and Kaltenbach 1961 and Lisoboa 1964). Quantitative determination of lovastatin was carried out by HPLC, using a 4-7 March 2007, Sharm El Sheikh, Egypt
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Merck-Lichrospher 60 RP select B reverse phase column with a UV detector (238 nm). A mobile phase mixture of trifluoroacetic acid (0.05%) and acetonitrile (55:45 and 67:33, v/v) at a flow rate of 1 ml was used. Production of Lovastatin on Semi-Industrial Scale: A 1.5 l B. Braun stirred tank (Biostat. ® A) fermentor (from B. Braun Biotech. International, Sortorius group, GmbH, Schwarzenberger, Germany) with one liter working volume was used in this study. The fermentor was equipped with pH, temperature, agitation, dissolved oxygen tension (DOT) and foam controllers. Seed cultures were carried out in 250 ml flask containing 50 ml of medium, held on a rotary shaker at 150 rpm, at 28 oC for 48 h. Seed culture flask (50 ml) for the fungal isolate (Penicillium camemberti No. 157) under study, which proved to be the higher lovastatin producer, was used to inoculate the fermentor at 30 oC. Fermentation lasted around 12 days. The culture medium contained grinding rice fragments (100 g/l) supplemented with 2.5 g/l of ammonium nitrate was used. The pH was adjusted to 5.5, temperature at 30oC, agitation at 400 rpm while the DOT in the culture broth was controlled via a sequential cascade control as air flow rate. The maximum and minimum set points of permitted airflow rates were 1.2 l/min and 0.1 l/min, respectively. The DOT during fermentation was controlled at medium (≃ 50%) of saturation. RESULTS AND DISCUSSION: Total of 75 different isolate of fungi were screened for their accumulation of lovastatin. About 57% of the isolates tested produced lovastatin of which 20, 16 and 7 isolates produced low (less than 150 mg/l), moderate (150 – 300 mg/l) and high levels (more than 300 mg/l) of this drug, respectively. Most of producer isolates belonging to the genera Aspergillus, Penicillium, Cladosporium, Humicola and Fusarium. Production of lovastatin was previously recorded by members of the genus Aspergillus (Alberts et al., 1980; Buckland et al., 1989; Manzoni et al., 1999; Safaa Mahmoud, 2001; Lai et al., 2002,2005; Lopez et al., 2003; Valera et al., 2005), Penicillium (Endo et al., 1976; Shindia, 1997; Safaa Mahmoud, 2001), Monoascus (Endo, 1985;Negishi et al., 1986), Trichoderma (Endo et al., 1986), Alternaria, Cochliobolus, Humicola, Stachybotrys and Paecilomyces (Murakawa et al., 1994; Safaa Mahmoud, 2001). Aspergillus was represented by 29 isolates (Table, 1). Emericella as Aspergillus related genus was represented by two isolates of E. nidulans. Twenty isolates ( 69 %) belonging to eight Aspergillus species(A. ochraceus, A. flavus, A. niger, A. wentti, A. fumigatus, A. terreus, A. ustus and A. sydowi) and one isolate (50 %) of E. nidulans had the ability to produce lovastatin. Low, 4-7 March 2007, Sharm El Sheikh, Egypt
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moderate and high levels of this drug was produced by 13, seven and one isolate, respectively, of the producer isolates belonging to the two genera (Table, 1). Nearly similar results were recorded by Safaa Mahmoud (2001). She examined the ability of 195 isolates belonging to 13 species and four species varieties of Aspergillus for statins production and found that 99 isolates of them ( 51 %) had the ability to produce statins. She recorded that the producer isolates belonging to Aspergillus flavus, A. fumigatus, A. nidulans var. dentatus (= Emericella nidulans var. dentate), A. niger, A. ochraceus, A. oryzae. A. sydowii, A. terreus, A. terreus var. africanus and A. ustus. Production of lovastatin by members of Aspergillus was previously recorded such as A. terreus (Alberts et al., 1980; Shindia, 1997; Szakacs et al., 1998; Kumar et al., 2000; Lai et al., 2002; 2005; Lopez et al., 2003), A. oryzae (Shindia, 1997; Saffa Mahmoud, 2001) and A. flavipes (Valera et al., 2005). On the other hand, this is the first report on the production of this drug by A. wentii. Table (1): Accumulation of lovastatin by different isolates belonging to various species and varieties of Aspergillus and Emericella. Organisms Genus: Aspergillus Subgenus: Circumdati Section: Circumdati A. ochraceus Wihelm Section: Flavi A. flavus Link A. flavus var. columnaris Raper & Fennell A. tamarii Kita Section: Nigri A. niger Van Tieghem Section: Wentii A. wentii Wehmer Subgenus: Fumigati Section: Fumagti A. fumgatus Fresenius Subgenus: Nidulantes Section: Terii A. terreus Thom Section: Usti A. ustus (Bain.) Thom & Church Section: Versicolores A. sydowi (Bain. & Sart.) Thom & Church Subgenus: Ornati Section: Oranti A. ornatus Raper, Fennell & Tresner Genus: Emericella E. nidulans (Eidam) Vuillemin Total
Code No.
Total Isolates Tested
(+) Ve isolates
(-) Ve isolates
Low
*
Moderate**
High***
101,106
2
1
1
-
-
16,20,13,33-35 37
6 1
1 1
2 -
3 -
-
129
1
1
-
-
-
83,88,93,98,99
5
-
5
-
-
145
1
-
1
-
-
68,73,74,76
4
1
2
1
-
132,138-141
5
2
1
1
1
142
1
-
-
1
-
127,128
2
1
-
1
-
1
-
-
-
114
1
149,150
2
1
1
-
-
-
31
10
13
7
1
*
Low levels of lovastatin = less than 150 mg/l medium ** Moderate levels of lovastatin = from 150-300 mg/l medium *** High levels of lovastatin = more than 300 mg/l medium
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Twenty three isolates of 15 species and one species variety of Penicillium belonging to four subgenera were tested in this study for respective abilities to produce lovastatin (Table, 2). Only 17 isolates ( 74 %) could produce lovastatin. Six, seven and four isolates had the ability to produce low, moderate and high levels of this drug, respectively. Penicillium capsulatum, P. spinulosum, P. funiculosum. P. albidium, P. brevicompactum, P. camemberti, P. chrysogenum, P. cyclopium, P. multicolor, P. nigricans and P. viridicatum had the ability to produce lovastatin. Four of them( P. brevicompactum, No. 155; P. camemberti No. 157, P. multicolor No. 221 and P. spinulosum No. 232 were recorded as high producers (produced more than 300 mg/l). Some members of the genus Penicillium were previously recorded as lovastatin producers of which P. citrinum, P. chrysogenum, P. aurantiogriseum, P. brevicompactum, P. corylophilum, P. digitatum, P. duclauxii and P. expansum (Endo et al., 1976; Lam et al., 1981; Hosobuchi et al., 1993; Shindia, 1997; Safaa Mahmoud, 2001). Production of lovastatin by the isolates of each of P. capsulatum, P. spinulosum, P. funiculosum, P. albidium, P. camemberti, P. cyclopium, P. multicolor, P. nigricans and P. viridicatum recorded in this study may be the first report on the production of this drug by these members of Penicillium. Eighteen isolates belonging to nine species of six genera representing two families of hyphomycetes in addition to three isolates of Mucor circinelloides (one isolate) and M. fuscus (two isolates) representing family Mucoraceae of Zygomycetes were tested for lovastatin production (Table, 3). Only five isolates belonging to three genera proved to be lovastatin producers. These isolates were Cladosporium cladosporioides (No. 243 & 244), Fusarium moniliforme (No. 276 & 277) and Humicola grisea (No. 246). Safaa Mahmoud (2001) recorded the production of statins by Humicola grisea. Also, several isolates belonging to various fungal genera and species other than members of Aspergillus and Penicillium were previously recorded by several workers as statin producers including: Alternaria alternata, Dormatmyces stemonitis, D. nanus, Gymnoascus umbrianus, Hyphomyces chrysospermus, Monoascus rubur, M. paxii, Ulocladium constiale, Stachybotrys chartarum, Trichoderma harzianum, T. pseudokoningi, T. longibrachiatum, Pacecilomyces varietti and Scopulariopsis brevicaulis (Endo, 1979;1980;1985; Endo et al., 1986; Murakawa et al., 1994; Manzoni et al., 1999; Safaa Mahmoud, 2001). According to literatures available this the first record for production of lovastatin by Cladosporium cladosporioides and Fusarium moniliforme.
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The seven isolates recorded as the higher lovastatin producers (Aspergillus terreus No. 141, Penicillium spinulosum No. 232, P. brevicompactum No. 155, P. camemberti No. 157, P. multicolor No. 221, Fusarium moniliforme No. 276 and Humicola grisea No. 246) were selected for comparison between static and shaking cultivation methods. Lovastatin levels produced by the seven isolates were ranged from 320 to 460 and 310 to 360 mg/l using shaking and static cultivation, respectively (Table, 4). The highest level of lovastatin (460 mg/l) was recorded by P. camemberti No. 157, followed by P. spinulosum No. 155 (430 mg/l) and Fusarium moniliforme No. 276 (410 mg/l) using shaking cultivation. Table (2): Accumulation of lovastatin by different isolates belonging to various species of Penicillium. Organisms
Subgenus: Aspergilloides P. capsulatum Raper & Fennell P. spinulosum Thom Subgenus: Biverticilium P. duclauxi Delacorix P. funiculosum Thom P. purpurogenum Stoll Subgenus: Furcatum P. corylophilum Dierckx Subgenus: Penicillium P. albidium Sopp P. brevicompactum Dierckx P. camemberti Thom P. chrysogenum Thom P. cyclopium Westling P. digitatum (Pers. Ex Fr.) Saccardo P. multicolor GrigorievaManoilova & Paradielove P. nigricans (Bain.) Thom P. verrucosum var. cyclopium (Westling) Samson, Stolk & Hadlok P. viridicatum Westling Total
Code No.
(+) Ve isolates
Total isolates Tested
(-) Ve isolate
Low*
Moderate**
High***
163
1
-
1
-
-
232
1
-
-
-
1
209 213 227
1 1 1
1 1
-
1 -
-
204
1
1
-
-
-
152 155 157,158 177,178,181,183 185,186 206 207,208
1 1 2 6
-
1 4
1 2
1 1 -
1 2
2
-
1 -
-
221
1
-
-
-
1
224 235
1 1
1
-
1 -
-
236 -
1 23
6
6
1 7
4
*
Low levels of lovastatin = less than 150 mg/l medium Moderate levels of lovastatin = from 150-300 mg/l medium *** High levels of lovastatin = more than 300 mg/l medium **
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Table (3): Accumulation of lovastatin by different isolates belonging to Hyphomycetes and Zygomycetes. Organisms
Group: Hyphomycetes Family: Dematiaceae Alternaria alternata (Fries.) Keister Cladosporium cladosporioides (Fries.) Devries Humicola grisea Traaen Stachybotrys chartarum (Ehrenberg) Hughes S. theobromae Hansf. Trichoderma hamatum (Bon.) Bainier T. koningii Oudemans T. polysporum (Link ex Pres.) Rifai Family: Tuberculariaceae Fusarium moiliforme Sheldon Group: Zygomycetes Family: Mucoraceae Mucor circinelloides van Tieghem M. fuscus Bainier Total
Code No.
(+) Ve isolates
Total isolates Tested
(-) Ve isolate
Low*
Moderate**
High***
239,241
2
2
-
-
-
243-245
3
1
1
1
-
246 251
1 1
1
-
-
1 -
253 255-257
1 3
1 3
-
-
-
258 260-262,264
1 4
1 4
-
-
-
276,277
2
-
-
1
1
320 329,331
1 2
1 2
-
-
-
-
21
16
1
2
2
*
Low levels of lovastatin = less than 150 mg/l medium Moderate levels of lovastatin = from 150-300 mg/l medium *** High levels of lovastatin = more than 300 mg/l medium **
Table (4): Comparison between surface (static) and submerged (shaking) cultivation for lovastatin production (mg/l) using the synthetic medium by the seven highly producer organisms. Organisms Aspergillus terreus Penicillium spinulosum P. brevicompactum P. camemberti P. multicolor Fusarium moniliforme Humicola grisea
Code No.
Static cultivation
Shaking cultivation
141 232 155 157 221 276 246
310 330 320 360 320 320 310
340 430 370 460 350 410 320
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Most of workers studied the production of lovastatin by filamentous fungi using shaking cultivation method and recorded levels of lovastatin formation nearly similar to those recorded in the present study. Safaa Mahmoud (2001) recorded that the maximum level of lovastatin produced by A. terreus No. 385, grew on modified Czapek’s medium contained 140 g glucose per liter at 28oC for 10 days, was 323 mg/l. Lai et al. (2002) studied the production of lovastatin by A. terreus ATCC 20542 in both shaking flask and 5 L fermentor cultivation and found that the level of lovastatin reached 510 mg/l medium after 10 days of incubation used shaking flasks cultivation. Also, Lai et al. (2005) found that the level of lovastatin by the same fungal isolate (A. terreus ATCC 20542) grown on another fermentation medium using shake flask cultivation reached 331 mg/l medium after 10 days of incubation. For optimization of fungal growth as well as maximization of lovastatin production, the superior fungal isolate (P. camemberti No. 157) was selected for a series of experiments to determine the role of some nutritional and environmental factors that influence production of lovastatin by this isolate. Maximum yield of lovastatin (520 mg/l) was obtained when 150 g/l glucose and 2.5 g/l ammonium nitrate were used as the sole carbon and nitrogen sources, respectively. The optimal pH, temperature and incubation period were 5.5, 30oC and 12 days, respectively. Safaa Mahmoud (2001) extensively studied the different nutritional and environmental factors influence the production of lovastatin by A. terreus No. 385, and reported that glucose (140 g/l) and ammonium nitrate (2.5-3.0 g/l) were the best carbon and nitrogen sources, respectively, which give the highest level of lovastatin. Also, she found that the optimum temperature, pH and incubation period for lovastatin production by A. terreus No. 385 were 30oC , pH 5-6 after 8-10 days of incubation, respectively. Hosobuchi et al. (1993) reported that nitrogen content from 0.3 % to 0.35% was suitable for compactin production by P. notatum. Shindia (1997) noted that the optimum temperature and pH for lovastatin production by A. terreus were 30oC and pH 5.5-6.0, respectively. Previous reports indicated that incubation period of 8-12 days were optima for lovastatin production by various fungal isolates (Endo, 1979; Shindia, 1997; Kumar et al., 2000; Lopez et al., 2003; Lai et al., 2005; Valera et al., 2005). In recent years, researchers have shown an increasing interest in the possibility to utilization of agro-industrial wastes or by-products as natural medium for microbial production of useful and economic products. In this investigation, an attempt has been made to investigate the possibility to utilization of some agro-industrial wastes and by-products: three kinds of 4-7 March 2007, Sharm El Sheikh, Egypt
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vegetables wastes (pea, kidney bean & mixed vegetables), wastes of other two vegetables used as pickles (carrot & turnip), four kinds of fruit wastes (apple, apricot, orange & peach), three industrial by-products (corn steep liquor, molasses & whey) and three agricultural by-products (wheat bran, rice husk & rice fragments) as natural medium for lovastatin production by the five high producer isolates (Penicillium spinulosum No. 232, P. brevicompactum No. 155, P. camemberti No. 157, P. multicolor No. 221 and Fusarium moniliforme No. 276). The five fungal isolates have the ability to grow and produce lovastatin, with variable levels, on the different three kinds of vegetable wastes but less than those recorded on the synthetic medium (Table, 5). The highest levels of this drug formed on kidney bean waste (370 mg/l) and mixed vegetable waste (280 mg/l) were produced by P. spinulosum No. 232 while the highest amount was recorded on pea waste (410 mg/l) and formed by P. camemberti No. 157. The highest amount of lovastatin formed on carrot waste was 410 mg/l produced by P. spinulosum No. 232 while all lovastatin levels produced by the five fungal isolates tested on turnip waste were negligible (Table, 5). The four kinds of fruit wastes media were suitable for fungal growth and lovastatin production by all fungal isolates examined (Table, 6). The highest levels of lovastatin recorded on apple (430 mg/l) and peach wastes (520 mg/l) were produced by P. camemberti No. 157 while those recorded on each of apricot (380 mg/l) and orange wastes (280 mg/l) were formed by P. multicolor No. 221. The three kinds of agriculture by-products media (wheat bran, rice fragment and rice husk) were recorded as highly favourable media for lovastatin production by P. camemberti No. 157 (Table, 7). The levels of lovastatin formed by this fungal isolate on each of the three media (510-520 mg/l) were nearly equal to those recorded by the same isolate on the optimized synthetic medium (520 mg/l). Also, the other four fungal isolates under study have the ability to grow well and produce considerable amount of lovastatin on the three by-products media (Table, 7). The five fungal isolates tested grew and produced relatively moderate levels (145-360 mg/l) of lovastatin on molasses medium while negligible results were recorded on each of corn steep liquor and whey media ( Table, 7 ). Several waste solid substrates and their combination were studied to determine its suitability for production of lovastatin. Valera et al. (2005) examined the possible utilization of wheat bran, bagasse, barley, grain bran, 4-7 March 2007, Sharm El Sheikh, Egypt
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soybean meal in addition to pineapple and orange fruit wastes for lovastatin production by A. flavipes BICC 5174. They found that wheat bran, barley and combination of the two substrates (1:1) were the most suitable substrate yielding 13.49, 9.67 and 11.70 mg of lovastatin per g of dry substrate, respectively. Also, they noticed that using fruit wastes (pineapple & orange) as substrates formed low levels of lovastatin. Table (5): Production of lovastatin (mg/l) by the selected five highly producer organisms grown on vegetable, pickle wastes as well as synthetic medium for 12 days as shaking cultivation. Fungal isolates Tested
Code No.
Synthetic medium
Control* Penicillium spinulosum P. brevicompactum P. camemberti P. multicolor Fusarium moniliforme
0 232 155 157 221 276
0 460 380 520 360 440
Kind of vegetable wastes Kidney Mixed Pea bean vegetable 0 0 0 380 370 280 310 215 170 410 .330 210 300 340 260 150 160 155
Kind pickles wastes carrot
turnip
0 410 140 0 380 280
0 75 120 105 85 110
Table (6): Production of lovastatin (mg/l) by the selected five highly producer organisms grown on fruits wastes as well as synthetic medium for 12 days as shaking cultivation. Fungal isolates tested Control* Penicillium spinulosum P. brevicompactum P. camemberti P. multicolor Fusarium moniliforme
Code No. 0 232 155 157 221 276
Kind of fruit wastes
Synthetic medium
Apple
Apricot
Orange
0 460 380 520 360 440
0 210 270 430 240 280
0 200 300 210 380 290
0 156 210 260 280 220
Peach 0 260 280 520 270 405
Table (7): Production of lovastatin (mg/l) by the selected five highly producer organisms grown on agriculture and industrial byproduct wastes as well as synthetic medium for 12 days as shaking cultivation. Kind of agriculture by-products
Fungal isolates tested
Code No.
Synthetic medium
Wheat bran
Rice fragment
Rice husk
Control* Penicillium spinulosum P. brevicompactum P. camemberti P. multicolor Fusarium moniliforme
0 232 155 157 221 276
0 460 380 520 360 440
0 200 310 510 360 190
0 180 240 520 290 280
0 220 380 520 280 270
Kind of industrial by-products Corn steep molasses whey liquor
0 60 0 120 120 80
0 145 260 310 360 240
0 65 0 110 0 70
*Control : Wastes or by-products without fungal inoculum
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El-Refai and El-kady (1969) and Ghanem et al. (1990) reported the possible utilization of molasses for sterols production by yeast and filamentous fungi, respectively. Kahraman and Yesilada (2001) used industrial and agricultural wastes as substrates for loccase production by Coriolus versicolor ATCC 200801 and Funalia trogii ATCC200800 as white rot fungi and recommended using of these waste in the production of important lignocellulolytic and other biotechnological enzymes, respectively. Sallam et al. (2003) used a medium composed of cane sugar molasses (3%) and corn steep liquor (1 %) for cyclosporin A production by A terreus and recorded the production of 45.23 mg cyclosporin A per one liter medium. More recently, Ragaa Kotby (2006) found that ten fungal isolates (one of each of A.ustus, Fusarium nivale, F. oxysporum, F. moniliforme, Trichoderma hamatum and T. pseudokoningii in addition to four isolates of T. harzianum) had the ability to grew well and produce cyclosporin A at levels fluctuated between 400 and 1200 µg/ 50 ml of 10% molasses medium. An attempt has been made for production of lovastatin by the superior producer isolate (P. camemberti No. 157) grown on the more favourable agricultural by-product medium (rice fragment medium) using a 1.5 liter laboratory fermentor. The results revealed that at the first 24 hours lovastatin produced was 210 mg/l, after this time, lovastatin level was increased gradually with the increasing of the fermentation period and maximum level (620 mg/l) was obtained at ninth and tenth days of incubation. Slight decrease of lovastatin level was recorded after this time of incubation (Table, 8). Nearly similar results were recorded by Lai et al. (2002 & 2005). They examined the lovastatin production by A. terreus ATCC 20542 in presence or absence of an oxygen carrier in both shaking flask and 5 l fermentor cultivations. They observed the formation of lovastatin in the 5 l fermentor (without added oxygen carries) at the first 24 hours and reaching maximum level (458 mg/l) at the tenth days. Also, they observed a slight decrease in lovastatin levels after the tenth days. On the other hand, Kumar et al. (2000) used batch hand fed-batch fermentations in 1000 l bioreactor for production of lovastatin by A. terreus DRCC 122. They found that the maximum levels of lovastatin were 1270 and 220 mg/l after 12 days of incubation using batch and fed-batch fermentations, respectively. Based on the present and other experimental observations, further study for regulation of lovastatin production by special nutritional, environmental and feeding strategies as well as selection and improvement of producer strain are need. 4-7 March 2007, Sharm El Sheikh, Egypt
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Table (8): Accumulation of lovastatin (mg/l) by highly producer isolate (Penicillium camemberti No. 157) grown on rice fragment medium for 12 days using a laboratory fermentor. Lovastatin concentration (mg/l)
Fermentation period (Days)
0.0
Control (zero) 1 2 3 4 5 6 7 8 9 10 11 12
210 320 460 500 540 580 600 600 620 620 560 520
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