Optimization of Banana Juice Fermentation for the Production of Microbial Oilt

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1988, p /88/ $02.00/0 Copyright 1988, American Society for Microbiology Vol. 54, No. 3 Optimization of Banana Juice Fermentation for the Production of Microbial Oilt ESTHER Z. VEGA, BONITA A. GLATZ,* AND EARL G. HAMMOND Department of Food Technology, Iowa State University, Ames, Iowa Received 14 September 1987/Accepted 21 December 1987 Apiotrichum curvatum ATCC (formerly Candida curvata D), a lipid-accumulating yeast, was grown in banana juice. The optimum conditions for biomass production in shake flasks were 30 C growth temperature, efficient aeration, a juice concentration of 25%, and preliminary heat treatment at less than sterilization conditions. Under controlled conditions in a fermentor, 20% banana juice was optimum. High concentrations of yeast extract (0.3%) increased biomass production by 40% but decreased oil production by 30%. A lower yeast extract concentration (0.05%) increased biomass production by 2% and oil production by 25%. The best growth and oil production were observed when asparagine (1.4 g/liter) and mineral salts were added to the banana juice. The addition of minerals seemed to improve the utilization of carbon. Growth inhibition was observed when the fermentor was aerated with pure oxygen, even when additional nutrients were present. A fed-batch process permitted the juice concentration to be increased from 15 to 82%; biomass accumulation was three times higher than in batch fermentations. However, the cellular lipid content was only 30% of dry weight, and chemical oxygen demand reduction was slow and inefficient. Fermentation of waste materials by oleaginous, or lipidproducing, microorganisms can have several beneficial effects. Low-value materials can be converted to higher-value end products such as single-cell oil and single-cell protein. The amount of organic matter in the waste material can be reduced to more manageable levels. Substrates rich in carbohydrate but low in nitrogen can be used. Work in microbial lipid production began in our laboratory as a means of utilizing whey, which is a by-product of the cheese industry. Four yeast strains able to grow on cheese whey and ultrafiltered whey permeate and to accumulate large quantities of lipid in the form of large intracellular oil droplets were isolated in our laboratory (9). The most efficient oil producer, Candida curvata D (reclassified as Apiotrichum curvatum ATCC 20509), has been shown to accumulate up to 60% of its dry weight as an intracellular oil droplet and to use up to 97% of the chemical oxygen demand (COD) of whey permeate (9). The oil produced is triglyceride with a fatty acid composition similar to that of cocoa butter. It is rich in oleic acid (51%) and contains significant amounts of palmitic (30%), stearic (11%), and linoleic (6%) acids (8). A. curvatum ATCC can grow on a wide range of carbohydrates. Glatz et al. (6), in shake flask experiments, demonstrated that this organism could use glucose, galactose, cellobiose, sucrose, and lactose as carbon sources. Evans and Ratledge (4) showed that, in batch culture, lactose supported the best biomass production and that xylose was best for lipid production. Lactose and xylose were also the best substrates for continuous culture. The ability of the organism to metabolize a wide variety of sugars suggested that other agricultural and food-processing wastes might be able to support growth and oil production. Oat hulls, bean blanch water, corncobs, wood cellulose, ripe bananas, and molasses were examined in our laboratory (7). These experiments showed that A. curvatum lacked the necessary enzymes to grow well on cellulosic materials, * Corresponding author. t Journal paper no. J of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA Project such as corncobs and oat hulls, but it grew well on materials rich in sugar, such as molasses and ripe bananas. Under controlled conditions in a fermentor, growth and lipid production were not very efficient on a molasses substrate. However, preliminary experiments indicated that ripe bananas were a good source of carbon and supported good growth and lipid production by A. curvatum. Banana production in many Latin American countries represents a major source of income. In 1982, the principal producer countries produced 41.5 million tons of bananas, of which only 17% were exported. The trend in the banana market for the last few years has been overproduction and little or no increase in demand. Because quality standards are high for exports of the fruit, between 10 and 20% of the total banana crop may be rejected annually (10). Rejected bananas present a severe disposal problem in producer countries and frequently contribute to land and water contamination if they are left to rot. The purpose of this research project was to optimize conditions for the fermentation of ripe bananas by A. curvatum for the production of lipid, as a means of producing a valuable product from a common waste material. MATERIALS AND METHODS Banana juice preparation. Banana juice was prepared by a method adapted from Viquez et al. (12). Ripe bananas (8 in the VonLoesecke scale [13]) were bought at a local supermarket. They were washed, sliced, and blanched in live steam for 20 min. After separation from the skin, the pulp was milled, cooled to room temperature, adjusted to ph 4.0 with 3 N HCl, and incubated with 0.05% (wt/wt) pectinase from Aspergillus niger (Sigma Chemical Co., St. Louis, Mo.) for 2 h in an agitated water bath at 25 C. The juice was separated from the residue by centrifugation at 27,000 x g for 20 min at 4 C. Juice was filtered through Whatman no. 40 filter paper and stored at -29 C until needed. Unless otherwise indicated, banana juice was always diluted to 20% of its initial concentration with distilled water. The ph was adjusted to 5.2, and the banana juice solution was sterilized in the autoclave.

2 VOL. 54, 1988 BANANA JUICE FERMENTATION FOR MICROBIAL OIL 749 Additional nutrients, when required, were prepared as stock solutions, adjusted to ph 5.2, sterilized, and added to the sterile banana juice medium to give the final desired concentrations. These nutrients included asparagine (final concentration, 1.4 g/liter), yeast extract (final concentration, 5 or 30 g/liter), and the following salts (all final concentrations given as milligrams per liter): MgSO4 7H20, 1,000; - FeCl3 6H20, 20; CaCl2. 2H20, 200; ZnSO4 7H20, 1; MnSO4. H20, 2; NaCI, 60; and CuS04 * 7H20, 0.1. Several chemical analyses were performed on the juice. Solids. The solids content was determined according to procedure (1). Sugars. Sucrose, glucose, and fructose concentrations were determined by enzymatic methods developed for food analysis (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). Lipids. The lipid content was determined by the solvent extraction method of Folch et al. (5). Nitrogen. The nitrogen content was determined by the micro-kjeldahl method by procedure (1). Copper selenite was used instead of mercuric oxide. Starch. Starch was determined by two methods. The first was a quantitative enzymatic method for starch in foodstuffs (Boehringer Mannheim Biochemicals) in which the starch present in the sample was first hydrolyzed to glucose. To remove preexisting glucose, samples were treated by passage through a membrane with a 10,000-dalton cutoff or with glucose oxidase and catalase. Starch was also qualitatively determined by reaction with an iodine solution (3). A solution of soluble starch was used as a blank. Ash. The ash content was determined by procedure (1) ṗh. ph determinations were made by using a ph meter (type PHM61a; Radiometer, Copenhagen, Denmark). Viscosity. The viscosity was determined at 25 C by using an Ostwald viscosimeter (Fisher Scientific Co., Chicago, Ill.). Organism. Candida curvata D, previously isolated by Moon et al. (9) and now reclassified as Apiotrichum curvatum ATCC 20509, was used in all experiments. Stock cultures were maintained on slants of YM agar at 4 C and were transferred monthly. YM agar contained (in grams per liter): peptone, 5; yeast extract, 3; malt extract, 3; glucose, 10; and agar, 20. Shake flask studies. Shake flask studies were performed to determine the environmental conditions and medium composition that would produce maximum cell dry weight. Unless otherwise indicated, cultures were grown in 250-ml Erlenmeyer flasks containing 25 ml of medium incubated at 30 C with shaking at 150 rpm in a forced-air incubator shaker (Lab-Line Instruments Inc., Melrose Park, Ill.). Closures for flasks were milk filters (Johnson and Johnson Co., Walpole, Mass.) to allow good gas exchange or Dispo plugs (American Scientific Products, McGaw Park, Ill.) for more restricted aeration. Culture growth was followed by determining dry weight. Statistical analysis. Experiments were organized as factorial designs (11) to optimize conditions with high efficiency. Treatments were selected according to a central composite second-order design (2) for experiment 1, a full factorial design for experiment 2, and a central composite secondorder rotatable design (2) for experiment 3. Values used for the variables under study in these experiments are presented in Tables 1, 2, and 3. Analysis of the results by response surface methods (2) was performed with an SAS software package (SAS Institute Inc., Cary, N.C.) on the Iowa State University computer. Fermentation conditions. Fermentations were run in a 500-ml working volume magnetically coupled bench top fermentor (Bioflo model C30; New Brunswick Scientific Co., New Brunswick, N.J.). Glass rods were substituted for the stainless steel baffles, and a Teflon stirring bar was used as the impeller. The ph was controlled by an automatic ph control system, model Ph 40 (New Brunswick). The airflow rate was controlled by a flow meter. Air entered the vessel through a sparger (Fisher). All fermentations were done at 30 C, with the ph maintained between 5.2 and 5.6. The aeration rate was 1 vol/vol per min, and the agitation rate was 150 to 200 rpm. Antifoam was added as required. The inoculum was grown for 18 h at 30 C with shaking in the same medium that was to be used in the fermentor. A suitable volume of inoculum was added to the fermentor to obtain an initial cell count of 106 cells per ml. Fed-batch fermentation. In this system, the juice concentration in the fermentor was increased slowly from 15 to 82% over 78 h by the addition of increasing amounts of pure sterile banana juice. The fermentor was first run as a batch system for 12 h with 15% banana juice as the growth medium. The following program of additions was followed: first four additions, 17.0 ml of pure banana juice; next four additions, 33.5 ml; last four additions, 50.0 ml. Growth was checked every 5 h by optical density (OD) measurements, and additions were made at these times unless the OD had not increased significantly since the previous addition. Samples were taken at periodic intervals for cell number, cell dry weight, COD, and nitrogen determinations. Lipid was extracted at 144 and 160 h. The fermentation was run for 160 h. Cell dry weight determinations. For each dry weight determination, a 5-ml culture sample was centrifuged in a tared tube at 3,638 x g for 10 min at room temperature and washed first with 10 ml and then with 5 ml of 0.1 M phosphate buffer at ph 7.0. The cell paste was dried at 65 C for 48 h in a vacuum oven. After cooling, the tube was weighed, and the cell dry weight (grams per liter) was calculated. Reported results are the mean values of two determinations. All other analyses were performed as described previously (9). Samples were taken from the fermentor at 72 and 96 h. All reported results are the average of two replicate samples. RESULTS AND DISCUSSION Characteristics of juice. A bright yellow juice was extracted from the pulp with a yield of 82% after incubation with the pectinase enzyme. The juice had a nice aroma, was clear, and had an absolute viscosity of 3.72 P. The juice became darker, and some cloudiness accompanied by precipitate formation was detected after sterilization in the autoclave. It is possible that the change in appearance was due to the formation or degradation of components by heat. The juice contained 24.4% (wt/wt) total sugars, including 5.2% glucose, 3.9% fructose, and 15.3% sucrose. Amounts of individual and total sugars increased by about 15% after sterilization. The juice also contained 0.62% lipid, 0.77% ash, and 0.087% nitrogen. The amount of nitrogen in the pulp was low (data not presented), and a very small amount was extracted into the juice. It is possible that the nitrogen is bound in some complex form that remains in the residue after juice extraction. Starch was not detected in the juice and was detected in the pulp only by the addition of iodine. Evidently, almost all the starch was converted to sugar in these

3 750 VEGA ET AL. TABLE 1. Values used for variables in shake flask experiment 1: four variables at five levels Level Juice Asparagine Starting Yeast extract (%) (mg/liter) ph (mg/liter) 2-< \ APPL. ENVIRON. MICROBIOL. TABLE 3. Values used for variables in shake flask experiment 3: two variables at five levels at two growth temperatures (30 and 34 C) Level Juice (%) Heating temp ( C) -V \ ripe fruits. Reports on the chemical composition of banana juice are lacking in the literature, but the composition of the banana pulp obtained in the current study agreed with that reported by others (14). Shake flask studies. The purpose of the shake flask experiments was to optimize cell dry weight production in banana juice by determining the best values for temperature, aeration, ph, additional nutrients, concentration of medium, and degree of heat treatment of the juice. Biomass rather than cellular lipid content was measured and optimized in these experiments because lipid analysis required considerable time and sample volume to perform and thus could not be used as a routine assay on large numbers of samples. The effects of and interaction among juice concentration, added asparagine or yeast extract, and starting ph were determined in experiment 1. Each variable was tested at five levels in a response surface treatment combination (Table 1). After 48 h, samples of 5 ml were taken for cell dry weight analysis. The factors that had the major effects (P < 0.05) on cell dry weight were juice concentration and yeast extract concentration. Regression analysis yielded the following quadratic equation: Y = x x 10-3 Xl x 10-6 X x 10-5 X x 10-8 X x 10-7 X1X2 where Y is the cell dry weight, X1 is the juice concentration, and X2 is the yeast extract concentration. The optimum juice concentration was 21%. The addition of yeast extract increased dry weight production only when lower juice concentrations were used. Over a range of juice concentrations from 19 to 23%, the addition of yeast extract did not improve dry weight production. This suggested that the juice contained sufficient nutrients for maximum growth in this concentration range. Growth was inhibited at higher concentrations ofjuice even when large amounts of yeast extract were added. In contrast, at juice concentrations below 19%, increasing amounts of yeast extract were necessary to support good growth. This indicated that some factor(s) became growth limiting at lower juice concentrations. The effects of and interaction among plug type (aeration), sterilization procedure, and concentration of asparagine, yeast extract, and juice were determined in experiment 2. Each variable was tested at two levels in a full factorial design (Table 2). Statistical analysis showed that the type of plug, the sterilization procedure, and the juice concentration were all significant parameters (P < 0.05), but the interactions among these were not important. Analysis of the data for these individual parameters in a general linear model procedure indicated that the greatest cell dry weights were obtained with milk filters as plugs (11.21 g/liter), with membrane filtration as the sterilization method (10.60 g/liter), and with 25% banana juice as the growth medium (10.39 g/liter). When these three conditions were present simultaneously, the cell dry weight was higher (12.63 g/liter) but was not significantly different. The addition of asparagine or yeast extract did not improve dry weight production. These results suggested that efficient aeration was necessary, that extensive heat treatment may either destroy a component that enhances growth or increase the production of an inhibitory factor(s), and that banana juice at the appropriate concentration contained sufficient nutrients for growth. The effects of and interaction between juice concentration and temperature of heat treatment were determined in experiment 3. Each variable was tested at five levels in a response surface treatment combination (Table 3). The design was run at two different growth temperatures (30 and 340C). Better growth was obtained at 30 than at 34 C. Statistical analysis showed that the temperature at which the juice was heated was the most significant factor and that the interaction between juice concentration and heat was also important. The quadratic equation derived from regression analysis was Y = X X2-8.3 x 10-5 X x 10-6 X x 10-5 X1X2 where Y is the cell dry weight, X1 is the juice concentration, and X2 is the temperature of heat treatment. As the juice concentration increased, maximal growth was obtained at lower temperatures. These results confirmed previous findings that heat treatment of the juice adversely affected culture growth, possibly because an inhibitory factor(s) may be generated in the juice by extensive heating. TABLE 2. Values used for variables in shake flask experiment 2: five variables at two levels Level Plug Asparagine Yeast extract (aeration) Sterilization procedure (mg/liter) (mg/liter) Juice (%) -1 Milk filter Autoclave Dispo plug Membrane filtration

4 VOL. 54, 1988 BANANA JUICE FERMENTATION FOR MICROBIAL OIL 751 TABLE 4. Performance of A. curvatum ATCC after 72 h in different concentrations of banana juice Banana juice Cell count Cell dry wt % COD concn (%) (log1o) (g/liter) reduction That better growth was obtained when mild heat treatment was used could have important economic implications. A. curvatum ATCC has been shown to compete well with other microorganisms in a fermentation process (8). If sterilization of the medium is not necessary for growth and production of oil by this microorganism, the cost reduction in a large-scale process could be significant. The optimum conditions determined in these experiments were 30 C growth temperature, efficient aeration, 25% juice concentration, and preliminary heat treatment at less than sterilization conditions. Experiments in the fermentor. The results obtained in the shake flask studies were applied to cultures grown under controlled conditions in the fermentor. Lipid production and COD reduction, as well as dry weight production, were measured to determine the success of each fermentation. A. curvatum was grown in three different concentrations of banana juice: 15, 20, and 25%. The organism grew during the first 48 h, after which cell mass and cell numbers were constant. A 20% concentration of banana juice supported the best growth (Table 4). In 25% juice, growth was poor. The small reduction in COD correlates with poor growth in 25% juice. Thus, even though shake flask experiments showed that 25% banana juice was optimum for culture growth, 20% juice was better in the fermentor. In an attempt to boost dry weight yield, two fermentations were run in 20% banana juice supplemented with 0.3 or 0.05% yeast extract. Results after 72 h are reported, but no changes were seen at 96 h (Table 5). The addition of yeast extract improved dry weight production. Carbon utilization as measured by COD reduction was also improved. Lipid production was favored with 0.05% yeast extract but was suppressed at the higher concentration of yeast extract. With the higher level of nitrogen provided by the yeast extract, the carbon-nitrogen ratio in this fermentation favored the production of total biomass at the expense of oil production. TABLE 5. Next, asparagine and mineral salts were tested to see whether they could mimic the stimulatory effect of yeast extract (Table 6). The addition of asparagine boosted cell numbers and cell dry weight, which was the expected result of providing an easily used nitrogen source. When only minerals were added, the results obtained were very similar to those obtained in unsupplemented banana juice, except that the percent reduction of COD was very high. The minerals seemed to improve the organism's efficiency of carbon utilization. Best results were obtained when both minerals and asparagine were added. Both carbon utilization and biomass production were stimulated, and very high lipid production was obtained. To study the effect of higher oxygen tension on growth and lipid production, pure oxygen was used to aerate a fermentation of 20% banana juice. Very poor growth was observed; only 2.4 g/liter (dry weight) was measured at 96 h. Microscopic examination revealed very few oil droplets in cells. In a second experiment in which oxygen was used to aerate a fermentation of 20% banana juice medium supplemented with asparagine and mineral salts, dry weight production was higher (4.25 g/liter) but, along with COD reduction, was very poor compared with a typical fermentation sparged with air. These results indicated that very high oxygen tension in the medium inhibits growth of and oil production by A. curvatum. It is possible that the organism needs a mixture of gases for growth and that the presence of only oxygen could cause the overproduction or accumulation of toxic compounds. Fed-batch fermentation. Results in the fermentor indicated that the substrate concentration should not exceed 20% juice for good dry weight and lipid production. In an attempt to increase fermentation efficiency by increasing substrate concentration, a fed-batch fermentation was tried. This system allows the culture to begin growth at a low substrate concentration but increases substrate concentration as culture density increases. The organism grew vigorously throughout the fed-batch process. Cell dry weights reached 30 g/liter, three times greater than in regular batch fermentations. The lipid content in the cells was only 30% of dry weight, but lipid production (9 g/liter) was greater than in batch fermentations. The percent reduction of COD was poor; only 32% of the COD was utilized over the 82-h period after the last addition of medium. At 160 h, the nitrogen content increased in the Effect of yeast extract on growth of and oil production by A. curvatum ATCC in 20% banana juice Yeast extract Cell count Cell dry wt % COD Oil produced % Oil per g concn (%) (log1o) (g/liter) reduction (g/liter) of dry wt TABLE 6. Effect of asparagine and salts mixture on growth of and oil production by A. curvatum ATCC in 20% banana juice after 96 h Medium supplement Cell count Cell dry wt % COD Oil produced % Oil per g (log1o) (g/liter) reduction (g/liter) of dry wt None Asparagine Salts Asparagine + salts

5 752 VEGA ET AL. medium, and a thin layer of material was recovered in the supernatant fraction after centrifugation of the broth. It seems that cell lysis occurred after 144 h. Probably the high levels of nutrients that allowed high cell mass production meant that nitrogen never became limiting to favor lipid production. The results of this fed-batch study indicate that this process may improve biomass production, but further work will be needed to optimize lipid production. LITERATURE CITED 1. Association of Official Analytical Chemists Official methods of analysis, 14th ed. Association of Official Analytical Chemists, Inc., Arlington, Va. 2. Cochran, W. G., and G. M. Cox Experimental designs. John Wiley & Sons, Inc., New York. 3. deman, J. M Principles of food chemistry. The AVI Publishing Company, Inc., Westport, Conn. 4. Evans, C. T., and C. Ratledge A comparison of the oleaginous yeast, Candida curvata, grown in different carbon sources in continuous and batch culture. Lipids 18: Folch, J., M. Lees, and G. S. Stanley A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: Glatz, B. A., E. G. Hammond, K. H. Hsu, L. Baehman, N. Bati, D. Brown, and M. Floetenmeyer Production and modifi- APPL. ENVIRON. MICROBIOL. cation of fats and oils by yeast fermentation, p In C. Ratledge, P. Danson, and J. Rattray (ed.), Biotechnology for the oils and fats industry. American Oil Chemists' Society, Champaign, Ill. 7. Glatz, B. A., M. D. Floetenmeyer, and E. G. Hammond Fermentation of bananas and other food wastes to produce microbial lipid. J. Food Prot. 48: Hammond, E. G., B. A. Glatz, Y. Choi, and M. T. Teasdale Oil production by Candida curvata and extraction, composition, and properties of the oil, p In E. H. Pryde, L. H. Princen, and K. D. Mukherjee (ed.), New sources of fats and oils. American Oil Chemists' Society, Champaign, Ill. 9. Moon, N. J., E. G. Hammond, and B. A. Glatz Conversion of cheese whey and whey permeate to oil and single cell protein. J. Dairy Sci. 61: Soto, M Bananas: cultivo y comercializacion. Litografia e Imprenta LIL, S.A., San Jose, Costa Rica. 11. Steel, R. G. D., and J. H. Torrie Principles and procedures of statistics. McGraw-Hill Book Co., New York. 12. Viquez, F., C. Lastreto, and R. D. Cooke A study of the production of clarified banana juice using pectinolytic enzymes. J. Food Technol. 16: VonLoesecke, H. W Bananas, 2nd ed. Interscience, New York. 14. Wills, R. B. H., J. S. K. Lim, and H. Greenfield Changes in chemical composition of "Cavendish" banana (Musa acuminata) during ripening. J. Food Biochem. 8: Downloaded from on January 27, 2019 by guest