Isolation and characterization of a ...

Isolation and characterization of a Saccharornycopsis lipolytica mutant showing increased production of citric acid from canola oil D AVID W. GOOD, RANDAL D RONIUK, G. ROSS LAWFORD,

AND JARED

E. FEIN'

Weston Research Centre, 1047 Yonge Street, Toronto, Ont., Canada M4W 2L3

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 05/03/13 For personal use only.

Accepted January 22, 1985

GOOD,D. W., R. DRONIUK, G. R. LAWFORD, and J. E. FEIN. 1985. lsolation and characterization of a Saccharomycopsis lipolytica mutant showing increased production of citric acid from canola oil. Can. J. Microbiol. 31: 436-440. A process for the production of citric acid from canola (rapeseed) oil using the yeast Saccharomycopsis lipolytica was examined. A citrate nonutilizing strain, designated NTG9, which had an improved citric to isocitric acid ratio, was isolated after mutagenesis of S. lipolytica ATCC 20228 with nitrosoguanidine. Although the mutant grew well on canola oil, unlike the parent strain or a spontaneous revertant (JF2), it did not grow on glycolytic intermediates below glycerate-3-phosphate, amino acids, hexadecane, or yellow kerosene. The mutant was shown to be impaired in the gluconwgenic pathway because of a loss of phosphoenolpymvate carboxykinase activity. A preliminary study of the effect of micronutrients on citric acid production by S. lipolytica NTG9 showed that manganese had a stimulatory effect on the process whereas zinc and iron were inhibitory. A revised growth medium was tested and found to increase citric acid production while decreasing that of iwcitric acid.

GOOD,D. W., R. DRONIUK, G. R. LAWFORD et J. E. FEIN. 1985. Isolation and characterization of a Saccharomycopsis

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lipolytica mutant showing increased production of citric acid from canola oil. Can. J. Microbiol. 31: 436-440. Une méthode de production d'acide citrique à partir de l'huile de canola (graine de Colza) à l'aide de la levure Saccharomycopsis lipolytica a été étudiée. Une souche désignée NGT9 qui n'utilisait pas de citrate et qui présentait un rapport acide citrique sur acide isocitrique amélioré a été isolée suite à la mutagenèse de S. lipolytica ATCC 20228 en présence de nitrosoguanidine. Bien que le mutant croîssait bien sur de l'huile canola, sa croîssance était affectée sur les intermédiaires glycolitiques inférieurs au glycérate-3-phosphate, sur les acides aminés, sur un hexadécane ou sur du kérosène jaune, contrairement à la souche parentale ou au mutant réverse spontané (JF2). 11 s'est avéré que le sentier gluconéogénique était altéré chez le mutant en raison d'une perte d'activité phosphoénolpymvate carboxykinase. Une étude préliminaire de l'effet des micronutriments sur la production d'acide citrique par S. lipolytica NTG9 a démontré que le manganèse avait un effet stimulateur sur le processus, tandis que le fer et le zinc étaient inhibiteurs. Un milieu de croissance a été modifié, testé et s'est avéré favoriser une augmentation de la production d'acide citrique ainsi qu'une diminution d'acide isocitrique. [Traduit par le journal]

Introduction The yeast Saccharomycopsis lipolytica can produce large quantities of citric acid with good yield when grown on a variety of fermentation feedstocks, including simple sugars, hydrocarbons such as n-paraffins and hexadecane, and numerous vegetable oils (Gutierrez and Erickson 1978; Souw et al. 1976; Txasawa and Takahashi 1981). Fermentation of sunflower, palm, olive, coconut, soybean, linseed, and rapeseed oils yielded between 48.8 and 155% (w/w) citric acid (Ikeno et al. 1975). Good yields of citric acid have also been reported from various fatty acids, the highest (107%, w/w) being achieved using oleic acid as the carbon source. However, little work has been done to optimize conditions of these fermentations. The use of hydrocarbons and n-paraffins as substrates for food-grade citric acid synthesis has been hindered by the potential carry-over of impurities such as carcinogenic polycyclic hydrocarbons in the final product (Fricke and Jenson 1975). Vegetable oils may prove to be a more suitable feedstock for citric acid production by S. lipolytica. The coproduction of isocitric acid, which has inferior buffering capacity and chelating ability, is a major drawback in using S. lipolytica as compared with Aspergillus niger for commercial citric acid production. However, S. lipolytica is able to utilize a wider range of carbon sources such as hydrocarbons than A. niger, which can be a major advantage, depending on the availability of feedstock. In S. lipolytica, the ratio and yield of citric and isocitric acids produced is both strain and carbonsource dependant (Glazunova and Finogenova 1976; Kozlova 'Author to whom reprint requests should be addressed.

et al. 1981). The ratio (citric: isocitric) can range between 1: 1 and 20: 1, as compared with the production of pure citric acid by A. niger. lmprovements on ratio in favour of citric acid have been attained by mutation and by medium optimization. Aconitasedeficient strains of S. lipolytica improved the citric acid yield and ratio on a glucose feedstock (Akiyama et al. 1972, 1973). Cultural conditions, specifically the presence or absence of various trace metals such as iron and rnanganese, have been shown to affect the ratio as well as yield of citric and isocitric acids on various feedstocks (Tabuchi et al. 1970; Hattori et al. 1974; Mandeva et al. 1981). The present research forms part of a study aimed at developing a commercial fermentation process for the production of citric acid from canola (rapeseed) oil using S. lipolytica. Since feedstock price is a major cost in fermentations, the option of using alternative substrates such as canola oil for the synthesis of citric acid would be desirable if this were economically advantageous. In the world marketplace, both sugar and vegetable prices have been subject to wide fluctuations over the past decade. Data will be reported concerning the isolation and biochemical characterization of an improved citric acid producing mutant strain derived from S. lipolytica ATCC 20228 as well as the results of a preliminary examination of the effect of several medium components on citrate production.

Materials and methods Cultures Saccharomycopsis lipolytica ATCC 20228, its derived mutant NTG9, and revertant JF2 were subcultured every 3 to 4 weeks on YM

GOOD ET AL

TABLE 1. Cornparison of citric acid production ability of S. lipolytica ATCC 20228 and mutant strain NTG9 after 9 days of batch fennentation


Strain

Isocitrate (g/L)

Total acid (g/L)

Y,

Y,,

(%)

(%)

N m : Yield of citric acid (YCA) and total acid (citnc plus isocitric) (Y,,) determined as grams of acid produced per 100 g of canola oil utilized.

.,

Time (hours)

FIG. 1. Citric and isocitric acid production by S. lipolytica ATCC 20228 in batch culture. NH,; X , canola oil; 0, biomass; a,citric isocitric acid. acid; 0, (Difco Laboratories) agar plates. They were lyophilized for long-term storage.

. . .............:... ........ .. .. .. ... .. .. .. .. .. . .: .: .: .. . .

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Citrate (g/L)

Product ratio (citric: isocitric)

1 j i ;

i

1 1 i

Media The chemically defined medium A was composed of the following (per litre): NH4CI, 1.6 g; KH2P04,1.75 g; K2HP04,0.49 g; Na2S04, 1.5 g; MgClz.6H20, 0.25 g; CaC12.2Hz0, 0.1 g; thiamine HCI, 200 kg; CaClz .2H20, 14.7 mg; FeC13 6HzO,24.4 mg; MnCI, .4Hz0,9.9 mg; ZnSO4-7H20, 7.2 mg; CoCI2.6H20, 2.38 mg; CuC12-2Hz0, 0.85 mg; H3BO3, 0.3 1 mg; Mo03, 1.44 mg. Medium B was the same as medium A with the following modifications: FeC12.6H20 and ZnS0,-7H20 were omitted and the concentration of MnC12.4Hz0 was increased to 39.5 mg/L. Canola oil was added to the medium at an initial level of 15 g/L. After 24-48 h, an additional 150 g/L of oil was added. Mutagenesis Mutagenesis of S. lipolytica ATCC 20228 by exposure to nitrosoguanidine (NTG) was performed by the method of Dawes et al. (1977) using 0.67% (w/v) yeast nitrogen base (YNB) (Difco Laboratories Ltd.) as the wash solution. Following mutagenesis, the cells were grown overnight in YNB containing 1% glucose, then streaked to produce isolated colonies on 1% glucose + YNB agar. Citrate nonutilizing mutants were identified by replica plating the colonies (Lederberg and Lederberg 1952) ont0 YNB agar containing citrate as the sole source of carbon.

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Growth conditions Batch fermentations were carried out in 2-L vessels (MultiGen F2000, New Brunswick Scientific). The pH was controlled at 5.0 during the growth phase and 3.5 during the subsequent citric acid production phase by automatic addition of 2 N KOH. Aeration was maintained at approximately 1 vvm (volume air/volume medium per minute). Agitation was maintained at 800 rpm using three angledblade impellers to keep the oil mixed with the liquid phase. Temperature was maintained at 30°C. Unless othenvise indicated, 1 .O L of medium A was inoculated with 20 mL of a 48-h shake-flask starter culture, which consisted of 100 mL of medium A in a 250-mL Erlenmeyer flask plus 0.5% CaCO, as a buffering agent. Preparation of ceil-free extracts Cells for the phosphoenolpyruvate carboxykinase (PEPCK) assay were grown as described above with 1% (w/v) glucose as the carbon source. For subsequent incubation with 2.0% (w/v) acetate, the cells were harvested by centrifugation in sterile centrifuge bottles at 4080 x g for 5 min, resuspended in sterile medium A, and recentrifuged. After a second resuspension in fresh medium A, the cells were transferred to a sterile fermentor vessel containing 1 .O L of medium A with 2.0% (w/v) sodium acetate as carbon source. The agitation, aeration, and temperature were unchanged. The pH was maintained at 6.0 by

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automatic addition of 2 N HCI. After 17 h incubation the cells were harvested by centrifugation. Cell-free extracts were prepared by washing the pellets twice in cold deionized water, twice in cold imidazole-HCI buffer (100 m M , pH 6.5), and homogenizing with a bench-top Braun homogenizer. The assay was performed on the supernatant obtained after centrifugation at 27 000 X g for 10 min. Assays Residual canola oil was measured by extraction of a IO-mL sample of the culture with hexane-chloroform (2: 1) followed by evaporation of the solvent and weighing of the residual solvent-extractable material. The aqueous phase was analyzed for citric and isocitric acids using Kits for Enzymatic Food Analysis (Boehringer-Mannheim, Canada Ltd.). In the citric acid assay (cat. No. 139 076), citrate was hydrolyzed to oxaloacetate and acetate by citrate lyase. Oxaloacetate was then reduced by NADH to L-malate in the presence of malate dehydrogenase. The oxidation of NADH was monitored at 340 nm in a Gilford Stasar II spectrophotometer and was stoichiometric with the amount of citric acid. The isocitrate assay (cat. No. 414 433) involved the conversion of isocitrate and NADP to a-ketoglutarate and NADPH by isocitrate dehydrogenase. The formation of NADPH was measured al 340 nm as before, and was stoichiometric with the amount of isocitric acid. In both cases the quantity of acid present in the sample could then be calculated from the measured change in absorption. Phosphoenolpyruvate carboxykinase (PEPCK) activity in the cell-free extracts was assayed by measuring the incorporation of I4CO2 into phosphoenolpyruvate in the presence of ADP to form [14C]oxaloacetate (Ballard and Hanson 1967).

Results and discussion Batch fermentation Figure 1 illustrates a typical time course of citric acid production by S. lipolytica ATCC 20228 from canola oil. It was found that the initial oil concentration in the fermentor had to be kept below 20 g/L to prevent the medium from inverting into a water-in-oil ernulsion ("butter phase") before the onset of growth of the yeast. This butter-phase formation typically retarded the duration of the fermentation by 12-24 h, corresponding to the tirne required to disperse the emulsion. This delay was presurnably caused by poor diffusion of nutrients and oxygen in the emulsion. An additional 150 g/L of oil could be added to the vessel after the initial phase of growth and dispersion of the ernulsion without reversion to the butter phase. Citric acid production began after depletion of the nitrogen source and the resultant cessation of growth, and ended after approxirnately 9 days total mnning time. The final product yieId of the fermentation is shown in Table 1. To obtain mutant strains having higher product yields and rates of productivity for citric acid, S. lipolytica ATCC 20228 was mutagenized with nitrosoguanidine. One isolate (NTG9), selected on the basis of its inability to utilize citric acid as the sole source of carbon, synthesized citric and isocitric acids

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CAN. J. MICROBIOL. VOL. 31, 1985

TABLE 2. Growth of S. lipolytica strains on various carbon sources


Substrate Fructose Glucose Glycerate Glycerol 2-p-Glycerate 3-p-Glycerate Canola oil Acetate Citrate Glutamate Isocitrate a-Ketoglutarate Malate Oxaloacetate Phosphoenolpyruvate Pyruvate Serine Succinate Hexadecane Yellow kerosene

Wild type 20228

Mutant NTG9

Revertant JF2

TABLE 3. Comparative activity of phosphoenolpyruvate carboxykinase in S. lipolytica ATCC 20228 and mutant NTG9

PEPCK activity (nmol COz.mg protein-' min-')

-

Condition

ATCC 20228

NTG9

Glucose Glucose shifted to acetate Acetate/glycerol

0.70

O"

5.86

O O

O

"No activity detected.

+ + + + + + + + + +

-

-

-

-

+ + + + + + + +

-

ND

-

ND

NOTE: Hexadecane, yellow kerosene, and canola oil were tested in liquid shake-flask cultures (medium A) at a level of 50 g/L. Al1 others were tested on a solid medium (yeast nitrogen base (Difco)), 1% (w/v) agar, 200 mg of the carbon source added to the centre of the plates and allowed to diffuse throughout the agar). ND. not detemlined.

from canola oil in batch culture at approximately the same overall yield as the parent strain, but with a significantly improved (by twofold) citric to isocitric acid ratio (Table 1). Mutant characterization To determine the nature of the lesion in strain NTG9. the growth patterns of the wild-type and mutant strains on a variety of carbon sources were examined (Table 2). The wild-type strain was able to grow on al1 of the compounds tested. In contrast, mutant NTG9 was unable to grow on any glycolytic intermediate below glycerate-3-phosphate or on any of the TCA cycle intermediates or amino acids tested. In shake-flask cultures, the mutant grew on canola oil but not on nhexadecane or yellow kerosene. A spontaneous revertant of strain NTG9, designated strain JF2, having the same carbon source utilization and fermentation charaiteristics as strain ATCC 20228, was isolated on plates containing citric acid as the sole carbon source. The isolation of this revertant indicated that the change in NTG9 probably involved a single mutation site. The carbon source utilization characteristics of strain NTG9 suggested that this isolate was deficient in the gluconeogenic pathway. This pathway is essentially a reversal of the Embden-Meyerhof pathway. Operation of the gluconeogenic pathway requires the presence of two unique enzymes: fructose diphosphatase di pho and phosphoenolpyÏuvate carboxykinase (PEPCK). Since strain NTG9 grew well on glycerol, FDP can be ruled out as the missing or defective enzyme. The ability of the mutant strain to grow on canola oil. butnot on hexadecane or kerosene, demonsiates the ability ofthe cells to use glycerol derived from the triacylglycerol fraction of the vegetable oil. If PEPCK was the site affected bv the mutation. mutant NTG9 would have been expected to grow on phosphoenolpyruvate and glycerate-2-phosphate. Owing to the instability of these

two compounds, however, it is possible that they were altered to forms nonmetabolizable b y the mutant ei'er before or during transport into the cell. Indirect supporting evidence for the theory that the site affected by the mutation in strain NTG9 could be PEPCK was provided by the work of Charpentier et al. (1976). They found that the addition of a variety of nitrogenous heterocyclic compounds such as 2-pyridine carboxylicacid ( ~ ~ i c o l i nacid) i c to cells of S. lipolytica strain IFP29 during the acid production phase increased the ratio of citrate to isocitrate synthesized from n-hexadecane. These compounds are known-to repress gluconeogenesis in animal cells by inhibiting PEPCK (DiTullio et al. 1974; MacDonald 1979). Direct assav of PEPCK activitv in the mutant was com~licated by the fact that this enzymé is inducible (Harasilta k d Oura 1975), requiring growth of the cells on a gluconeogenic substrate, none of which will support growth of the mutant. The rationale used by Flavell and Fincham (1968) for Neurospora crassa and by Armitt et al. (1976) for Aspergillus nidulans to assay for PEPCK activity in PEPCK-deficient mutants was adopted. Cells were grown on 1.5% (w/v) glucose, harvested, washed, resuspended in fresh medium containing 1.5% (w/v) acetate, incubated for 17 h, and assayed as described in Materials and methods. Cells of strain ATCC 20228 grown on glucose alone had some detectable enzyme activity (0.70 nmol mg protein-'. min-'). When grown on glucose and then shifted to acetate-containing medium, an eightfold increase in PEPCK activity was observed (Table 3). The mutant strain NTG9 had no detectable activity when grown under either of these conditions. Owing to the high affinity of S. lipolytica for canola oil, cell-free extracts of oil cells could not be prepared without the use of disrupting solvents. Consequently the assays were performed on extracts prepared from cells grown on a mixture of acetate and glycerol (95 : 5 ) , the basic precursor units of triacylglycerol. It was not possible to grow the microorganisms on a mixture of representative fatty acids and glycerol because of the toxicity of the former. No activity was detected in either the wild type or the mutant (Table 3), suggesting that the glycerol repressed formation of PEPCK. The mechanism of alteration of the citrate to isocitrate ratio by a PEPCK mutation in NTG9 is not clear. If the activity of PEPCK is repressed in wild-type cells grown on canola oil, as suggested by the data for cells grown on acetate-glycerol mixtures in Table 3, then these cells should be phenotypically similar to the mutant NTG9 with respect to PEPCK activity. The yield of citric plus isocitric acids from canola oil was found to be similar in the parent and mutant strains (Table l), in-

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439

GOOD ET AL.


dicating there is no difference in carbon flow into the citric acid cycle. Not enough is known about PEPCK activity in S. lipolytica and its interaction with the citric acid cycle enzymes and their equilibria to explain these results. Medium development Medium composition is known to influence the production of citric acid in both S. lipolytica and A. niger. Tabuchi et al. (1970) found that, in their strain of S. lipolytica growing on n-hexadecane, both the final concentration of total acid and the citrate to isocitrate ratio were optimum at 0.3% (w/v) NH4Cl. The presence of iron produced a negative effect on the citric to isocitric acid ratio. This cation is required for the activity of aconitase, therefore it influences the citrate to isocitrate equilibrium. Hattori et al. (1974) reported a similar negative effect for iron as well as zinc on citrate production by Candida zeylanoides using n-paraffins as carbon source. In A. niger, manganese inhibited the production of citric acid from sucrose (Rohr and Kubicek 1981). When manganese was limited, the intracellular ammonium pool increased and negated the feedback inhibition of phosphofmctokinase by citrate and ATP. This permitted an unregulated flow of carbon to be channelled from glucose to citrate. The effect of a variety of medium components on citric acid synthesis by S. lipolytica NTG9 in batch culture using canola oil as the carbon source were investigated in shake-flask cultures. Iron and zinc were found to have negative effects on yield of citric acid from canola oil, while manganese stimulated citrate synthesis (data not shown). The fermentation medium was revised (medium B) to incorporate the modifications suggested by the shake-flask experiments. A comparison of medium A and B is given in Table 4. Medium B was better than medium A, in terms of both product yield and ratio of citric to isocitric acids, throughout duplicate 9-day fermentations. Compared with MultiGen fermentors, citric acid production in shake flasks was poor and ceased after only 8 days of incubation. This was attributed to the lack of pH control, poorer aeration, and less effective mechanical dispersion of oil droplets. Aeration especially would be expected to be critical in the synthesis of an oxidized molecule such as citric acid from highly reduced triacylglycerols. A combination of mutagenesis and medium development have been shown to be complementary methods for improvement of citric acid production from vegetable oils such as canola oil by S. lipolytica. Mutagenesis, as also demonstrated by Akiyama et al. (1972, 1973), is a preferable way of influencing metabolic controls to produce desired effects, as opposed to the use of costly chemical agents such as 2-picolinic acid or the toxic agent fluoroacetate. The inclusion of such additives would be unacceptable for the production of foodgrade citric acid. Further improvements in product yield, concentration, and ratio, desirable to reduce recovery costs, will have to be made to this process. Since substrate costs can account for a major part of the price of a fermentation product (Righelato 1980), small increases in yield can significantly influence commercial feasibility. In our experiments the total yield of citrate and isocitrate from canola oil was about 140% (w/w). Since the maximum theoretical yield of citric acid from canola oil is about 200% (w/w), major increases in citrate yield are probably to be gained at the expense of isocitrate and through further process refinements. The major technical obstacle to commercial feasibility of citric acid production from canola oil by S.

TABLE 4. Effect of revised medium on production of citric acid by S. lipolytica NTG9 after 9 days culture in a NBS MultiGen fermentor

Medium

Citrate lsocitrate Total acid (g/L) (g/L) (g/L)

Product ratio (citric: isocitric)

Y,,

Y,,

(%)

(%)

NOTE: Y,, and Y,, were determined as grams of acid produced pet 100 g of canola oil utilized.

lipolytica would be the long fermentation time, resulting in a decreased fermentor productivity. Aspergillus niger can produce citric acid from molasses at a rate of 0.8 g -L-'- h-' with a yield approaching 90% of theoretical (Rohr and Kubicek 1981). The S. lipolytica process will have to be accelerated considerably from its present 9-day duration to compete with the established technology.

Acknowledgements We would like to thank Dr. B. H. Robinson, Department of Biochemistry, University of Toronto and Hospital for Sick Children, Toronto, Ont., for his generous assistance in conducting the PEPCK enzyme assays reported, and Dr. M. Beavan for his critical reading of the manuscript. This work was supported in part through an Industrial Research Assistance Program (IRAP) grant provided by the National Research Council of Canada to George Weston Ltd. AKIYAMA, S., T. SUZUKI, Y. SUMINO, Y. NAKOA, and H. FUKUDA. 1972. Production of citric acid from n-paraffins by fluoroacetatesensitive mutant strains of Candida lipolytica. Ferment. Technol. Today, Proc. Int. Ferment. Symp., 4th, 1972. pp. 613-617. 1973. Induction and citric acid productivity of fluoracetatesensitive mutant strains of Candida lipolytica. Agric. Biol. Chem. 37: 879-884. ARMITT,S., W. MCCULLOUGH, and C. F. ROBERTS. 1976. Analysis of acetate non-utilizing (acu) mutants in Aspergillus nidulans. J. Gen. Microbiol. 92: 263-282. BALLARD, F. J., and R. W. HANSON.1967. Phosphoenolpymvate carboxykinase and pyruvate carboxylase in developing rat liver. Biochem. J. 104: 866. CHARPENTIER, J. M., G. GLICKMAN, and P. MALDONADO. 1976. Process for producing citric acid by fermentation. U.S. patent 3 966 553. DAWES, 1. W., D. A. MACKINNON, D. E. BALL, 1. D. HARDIE, D. M. SWEET, F. M. ROSS, and F. MACDONALD. 1977. Identifying sites of simultaneous DNA replication in eucaryotes by N-methylNt-nitro-N-nitrosoguanidinemultiple mutagenesis. Mol. Gen. Genet. 152: 53-57. DITULLIO,N. W., C. E. BERKOFF, B. BLANK,V. KOSTOS, E. J. STACK, and H. L. SAUNDERS. 1974. 3-Mercaptopicolinic acid, an inhibitor of gluconeogenesis. Biochem. J. 138: 387-394. FLAVELL, R. B., and J. R. S. FINCHAM. 1968. Acetate non-utilizing mutants of Neurospora crassa. 11. Biochemical deficiencies and the roles of certain enzymes. J. Bacteriol. 95: 1063-1068. FRICKE,H., and S. B. JENSEN. 1975. Citric acid-A versatile food additive. Food Process. Ind. 36: 37-44. GLAZUNOVA, L. M., and T. V. FINOGENOVA. 1976. Enzyme activity of citrate, glyoxylate, and pentose phosphate cycles during synthesis of citric acid by Candida lipolytica. Mikrobiologiya, 45: 444-449. GUTIERREZ, J. R., and L. E. ERICKSON. 1978. Continuous culture of Candida lipolytica on n-hexadecane. Biotechnol. Bioeng. 20:

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