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An Anionic Galactomannan Polysaccharide Gum from a Newly-Isolated Lactose-Utilizing Bacterium. 11. Fermentation Kinetics and Lactose Transport James H,Flatt,t Timothy A. Cooper, Juan M. GonzalezJ Dina E. Dogger, Edwin N,Lightfoot, and Douglas C. Cameron* Department of Chemical Engineering, University of Wisconsin, 1415 Johnson Drive, Madison, Wisconsin 53706-1691
The facultative bacterium Erwinia ATCC 55046, isolated from the soil of a wheytreated farm field, produces an anionic galactomannan polysaccharide gum (designated lactan gum) from lactose and other sugars. On lactose semidefined medium in a 5-L fermentor, the organism had a maximum growth rate of 0.66 h-l and produced 26 g/L polysaccharide with a yield on lactose of 0.66 g/g and a volumetric productivity of 0.6 g/(L-h). In shake flasks, the best final gum concentration was obtained with a carbon t o nitrogen ratio of 4 0 1 (w/w). Gum yield and concentration was greater on disaccharides than on monosaccharides. The organism was found to transport lactose by a permease system as opposed t o a phosphotransferase system. Lactan gum can be produced efficiently from lactose, the major sugar in whey and whey permeate.
Introduction The utilization of lactose in whey permeate, a waste product of the dairy industry, is a significant worldwide economic and environmental problem. Implementing solutions to this problem have been difficult due to unfavorable economics resulting from the dilute nature of lactose in whey permeate (ca. 5% by weight) and the unavailability of large quantities of whey permeate at a single site. Recently, the price of lactose has risen dramatically. However, it is thought that whey utilization will continue to be a problem (Horton, 1992). It was noted in the preceding paper (Flatt et al., 1992) that polysaccharide gums are economically attractive products from lactose in whey permeate. One organism, tentatively identified as Erwinia sp. ATCC 55046, isolated from a farm field in Wisconsin, produces an anionic galactomannan gum (lactan gum) from lactose and other sugars with several potentially useful properties (Flatt et al., 1992).In this article, we present results on the influence of sugar type and the carbon to nitrogen ratio on lactan gum production. We also report the kinetics of the lactan gum fermentation on lactose. Finally, we describe the mechanism of lactose transport by Erwinia sp. ATCC 55046. The work reported here provides the basis for further process optimization of lactan gum production and for more detailed metabolic analysis and modeling. Materials and Methods Cell Culture and Maintenance. Erwinia sp. ATCC 55046 (American Type Culture Collection, Rockville, MD) was isolated from a Wisconsin farm field as described in the preceding paper (Flatt et al., 1992). The 5-L fermentation was conducted on lactose semidefined medium (LSM)at pH 7.0 as previouslydescribed (Flatt et al., 1992). Shake flasks were each inoculated with a single colony from a Petri dish and were incubated at 26 "C. The pH in the shake flasks was initially 7.0 and was not controlled. Shake-flask fermentations for the investigation of the
* Author to whom correspondence should be addressed. t
Current address: Kelco, Division of Merck, San Diego, CA 92113. Current address: Pillsbury, St. Louis Park, MN 55416. 8756-7938/92/3008-0335$03.00/0
carbon to nitrogen ratio were done in 250-mL cornerbaffled flasks containing 70 mL of the appropriate medium and were agitated on a rotary shaker at 175 rpm for six days. Shake-flaskfermentations for the sugar study used LSM or LSM in which the lactose was replaced with an equal amount (weight basis) of another sugar. For each sugar, four corner-baffled flasks were used three l-L flasks with 300 mL of medium and one 500-mL flask with 200 mL of medium. The cultures were agitated at 200 rpm (240 rpm after 40 h) on a rotary shaker for four days. Analytical Methods. CeZZ Mass. Cell mass was estimated by measuring the optical density at 660 nm. Dry cell weight, measured after the separation of polysaccharides and drying, was linearly related to the optical density at 660 nm, with an extinction coefficient of 7.4 L/(g.cm). To ensure measurement in the linear region of the spectrophotometer, samples were diluted to achieve an optical density of less than 0.15 absorbance units (au). Purified lactan gum, at the concentrations found in the fermentations, was found to contribute relatively little to the total absorbancea t 660 nm. The extinction coefficient of the purified gum at 660 nm was 0.061 L/(g.cm). Sugars and Fermentation Byproducts. The amount and type of sugars present in the broth were determined by HPLC (Waters Model 600; Waters, Milford, MA). Samples for analysis were prepared by removing the cells by centrifugation at 45000g for 30 min. If necessary due to high brothviscosity, the samples were diluted with water before centrifugation. After centrifugation, the supernatant was filtered through a 0.2-pm filter. Sugars were separated using a cation-exchange column in the Ca2+form (Sugar-PAK 11;Waters). The separation conditions were the following: mobile phase, deionized water; flow rate, 0.5 mL/min; sample injection volume, 10 pL; column temperature, 90 "C; and detector, refractive index maintained at 35 "C. The concentration of each sugar was determined by comparison of the peak areas with those of authentic standards. Fermentation byproducts, including organic acids, alcohols, and polyols in the supernatant were separated using a cation-exchangecolumn in the proton (H+)form (HPX87H; Bio-Rad Laboratories, Richmond, CAI. The sepa-
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ration conditions were the following: mobile phase, 0.01 N H2S04;flow rate, 0.5 mL/min; sample injection volume, 1OpL;column temperature, 40 "C; and detector, refractive index maintained a t 35 "C. Peak areas were compared with those of authentic standards. Carbon Dioxide. The carbon dioxide content of the fermentation outlet gas was determined by gas chromatographic analysis using a 6.5-ft-length column containing molecular sieve silica gel (801100mesh; Alltech Associates, Deerfield, IL). The carrier gas was helium (30 mlimin), and the column temperature was 60 "C. Carbon dioxide was detected by a thermal conductivity detector. The inlet and detector temperatures were 110 "C. The carbon dioxide production rate was determined from a 1.0-mL sample taken from the outlet gas with a flow rate of 4 standard Limin. Polysaccharide Gum Concentration. The gum concentration was estimated by gravimetric measurement of the precipitate formed when 2-propanol was added to the broth to achieve a final concentration of 75% (viv) 2propanol. A final 2-propanol concentration of 75 % gave complete gum precipitation with minimal coprecipitation of sugars and inorganic salts. A variable amount of broth, usually 20-50 mL, was used in the assay to increase the sensitivity of the test. To reduce the lactose coprecipitate, fermentation samples taken at the beginning of the fermentation, when the lactose concentration was high, were dialyzed against 10 volumes of deionized water for 48 h using rinsed Spectra/Por 2 membrane dialysis tubing (Spectrum Medical Industries, Los Angeles, CAI, having a molecular weight cutoff of 12 000-14 000. The dialysis water was changed daily (for the shake-flask sugar experiment the water was changed twice each day). The gum was precipitated from the dialyzed samples by the addition of sufficient 2-propanol to achieve a final concentration of 90 % (v/v). For all samples, solutions of broth and 2-propanol were held at 4 "C for 24 h before coarse filtration with preweighed and dried Whatman no. 2 paper. The filter paper was dried a t 105 "C until a constant weight was achieved. The net weight of precipitated material was corrected for the presence of cells by subtracting the portion due to cellular material, as estimated by optical density at 660 nm. Polysaccharide Gum Viscosity. Steady shear viscosity was determined using a Wells-Brookfield RV Cone and Plate Viscometer (Brookfield Engineering Laboratories, Stoughton,MA) as described in the precedingpaper (Flatt et al., 1992). Sugar Transport Mechanism Studies. The mechanisms of transport of lactose from the medium into the organism (ATCC 55046) were determined by classical biochemical techniques. The basis for differentiating between a permease or phosphotransferase mechanism involves identifying the type of @-galactosidasepresent in the cell (McKay, 1983; Saier, 1977; Kornberg, 1976). The presence of @-galactosidaseindicates the presence of intracellular lactose, which, in turn, indicates the presence of a permease-type transport. The presence of phosphop-galactosidase indicates the presence of intracellular lactose 6-phosphate,which, in turn, indicates the presence of phosphotransferase transport. The permease product (@-lactose)is hydrolyzed into @-glucoseand @-galactose by @-galactosidase,whereas lactose 6-phosphate (thephosphotransferase system product) is hydrolyzed by phospho0-galactosidase into galactose-6-P and glucose (McKay, 1983). o-Nitrophenyl galactoside (ONPG) and o-nitrophenyl @-D-galactopyranoside6-phosphate (ONPG-6-P)are non-
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metabolizable analogs of lactose and lactose 6-phosphate, respectively, which enable ready spectrophotometric determination of the type of @-galactosidasepresent in the cell. @-Galactosidasewill hydrolyze ONPG into galactose and o-nitrophenol, a yellow chromophore which has an absorption maximum at 420 nm. Phospho-@-galactosidase will hydrolyze ONPG-6-Pinto galactose 6-phosphate and o-nitrophenol. The enzymes have a high degree of specificity; hence, @-galactosidase will not hydrolyze ONPG-6-P and phospho-@-galactosidasewill not hydrolyze ONPG to any appreciable extent. A total of 250 mL of a mid-exponential-phase culture was prepared as described above. The cells were starved by the following procedure to increase activity of the sugar transport systems. A total of 50 mL of culture was centrifuged at 3000g for 10 min at 4 "C and the cell pellet was resuspended in an equal volume of phosphate buffer (pH 7.0) consisting of 6.10 g/L K2HP04, 3.05 g/L KH2PO4, and 0.1 g/L MgCl2 (the magnesium is necessary for @-galactosidaseactivity). The suspension was then recentrifuged to pellet the cells. This washing procedure was repeated twice prior to the final resuspension of the cells in phosphate buffer to achieve an optical density at 660 nm of 5.0 au. The cells were then incubated for 24 h in phosphate buffer prior to sugar uptake studies. Starved cells were also used to prepare toluene-treated cells and cell extracts. A toluene-treated cell suspension was prepared by adding one drop of a mixture of 90% acetone and 10% (v/v) toluene to 1 mL of whole cell suspension and incubating for 5 min at 37 "C. A cell extract suspension was prepared by centrifuging 30 mL of whole cell suspension at 3000g for 10 min and resuspending the cell pellet in 6 mL of sonication buffer containing 10 mM TrisqHCl (pH 8.01, 10 mM NaC1, and 1 mM sodium citrate. The suspension was cooled to 4 "C and sonicated for 15 min using a l-s cycle, 50% cycle duty, and 100 W of power (Heat System Ultrasonics, Inc., Farmingdale, NY). Microscopic observation indicated that the cells were completely disintegrated after this treatment. The extract was centrifuged at 12000g for 15 min at 4 "C to remove cellular debris. The clear supernatant contained the watersoluble enzymes of interest for this study. ONPG Test. A total of 1 mL of sample (whole cell, toluene-treated cell, or cell extract suspension) was added to 1mL of potassium phosphate buffer (pH 7.0) containing 0.1 g1L MgCl2 in a polystyrene spectrophotometer cuvette. The mixture was immediately placed in a spectrophotometer and the absorption at 420 nm was measured versus time at 25 "C until a constant value was achieved. A total of 1mL of ONPG (Sigma Chemical Co., St. Louis, MO) solution (0.01 M ONPG in the above phosphate buffer) was rapidly added to the cuvette and the solution was mixed thoroughly. The absorption a t 420 nm was measured versus time on a strip chart recorder. The ONPG-6-P assay was performed similarly; however, only 0.2 mL of ONPG-6-P (Sigma Chemical Co.) solution (0.012 M ONPG-6-P) was used. The rate of absorption change represents the rate of ONPG or ONPG-6-P hydrolysis per volume of sample. The absorbance change was converted to micromoles of ONPG or ONPG-6-P hydrolyzed by interpolating a standard curve of the absorbance versus o-nitrophenol (ONP) concentration. One absorbance unit corresponded to 0.46 pmol/mL ONP. The hydrolysis values were converted to the cell-specific turnover rate by accounting for the dry cell mass in the cuvette (an optical density reading at 660 nm of 1.0 au corresponded to 0.5 g of dried cells/L).
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Table I. Lactan Gum Production from Carbohydratess carbon source gum concn, g/L gum yield, g/g of sugar used broth viscosity, mPes @ 10 s-1 sucrose 10.3f 0.5 0.23 f 0.01 378 f 44 0.23 0.03 lactose 8.9 0.9 672 f 231 0.21 f 0.03 maltose 8.7 f 0.9 607 f 141 galactose 2.5f 0.5 0.17 f 0.01 57 f 30 fructose 1.9 f 0.7 0.05 0.02 15 f 8 mannose 1.1 f 0.1 0.05 f 0.01 6fl glucose 0.4f 0.1 0.02 0.005 3fO Resulta are the average of four replicates f one standard deviation (for pH only one replicate was measured).
*
Results and Discussion Fermentation Media Studiesand Kinetics. Screening of Carbohydrate Substrates. Erwinia sp. ATCC 55046 was grown on a variety of mono- and disaccharides (45g/L) in shake flasks to determine the best carbohydrate source for polysaccharide production. The only variable in this experiment was the type of carbon source. The results of this experiment (summarized in Table I) show that gum concentration was more than three times as high on disaccharides as on monosaccharides. The concentration of gum was similar (within the experimental variation) for all the disaccharides tested. The yield of gum was also higher on disaccharides than on monosaccharides. The final broth viscosity was greatest for lactose and maltose (the viscosity of purified gums from the flasks was not measured). The initial pH in all flasks was 7.0. The final pH was lower for the monosaccharides than for the disaccharides, indicating a greater production of organic acids. The difference in acid production on glucose and lactose was explored quantitatively in a three-replicate shake-flask experiment. After 24 h, the average total organic acid concentration of the glucose flasks was 3.2 mM, versus an average of 0.40 mM for the lactose flasks. Acetate was the primary acid present in both flasks. The average pH of the broth in the glucose flasks was 3.2 versus 3.9 in the lactose flasks. Little change in the total organic acid concentration or pH was observed in any of the flasks after 24 h. The above results suggest that disaccharides such as lactose are the preferred carbon source for lactan gum production. However, due to the pH variations and the difficulty of achieving good oxygen transfer in shake flasks, experiments in well-controlled fermentors are needed to fully determine the influence of sugars on gum production. Determination of the Optimal Carbon to Nitrogen Ratio. The yield of microbial polysaccharide gums is known to be enhanced by limiting a non-carbon element such as nitrogen or sulfur (Pace, 1987). The C to N ratio of whey permeate is approximately 50 (w/w) (Flatt, 1990). A shake-flask fermentation study with five replicates per C:N level was conducted to estimate the optimal C:N ratio for gum production. C:N ratios of 6,10,20,40,60,80, and 100 (w/w) were achieved by maintaining the lactose a t 45 g/L and the yeast extract at 1.8g/L and varying the amount of (NH&$304 added [the amount of carbon and nitrogen available from the yeast extract was assumed to be 0.52 g/L and 0.14 g/L, respectively (Zabriskie et al., 198211. The fermentations were run for 144h a t 26 "C. The results, summarized in Figure 1, indicate that a C:N ratio of approximately 40 is optimal. The gum concentration versus the C:N ratio was binodal, with an increase in gum concentration at C:N values from 80 to 100. The pH was not controlled in this study. The pH of the flasks with C:N ratios of 100 had an average value of 3.7 after 4 days and 3.5 after 6 days. From this experiment, a C:N ratio of 40 was selected for use in further studies. However, as
final broth DH 4.8 4.0 4.2 3.3 3.8 3.1 3.1
c 0
20
40
60
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CarbonfNitrogen Ratio, glg Figure 1. Lactan gum and cell production as a function of carbon to nitrogen ratio in shake flasks. The values are the average of five flasks; the standard error of the mean of gum concentration is 0.4 g/L and the standard error of the mean of cell mass is 0.1 g/L.
with the sugar study, experiments carried out in a wellcontrolled fermentor are needed for more rigorousprocess optimization. Fermentation Kinetics on Lactose. In order to study lactan gum production under more controlled conditions than could be achieved in shake flasks, Erwinia sp. ATCC 55046 was grown in a 5-L bioreactor with a 4.2-L working volume. The aeration rate was 1VVM (volume of air per volume of liquid per minute), and the agitation speed was 800 rpm. The fermentor temperature was controlled at 26 "C, and the pH was controlled at 7.0 by the addition of NaOH. The growth medium (LSM) had a C:N ratio of 40 (w/w). The fermentation results are summarized in Figure 2 and Table 11. The maximum growth rate was 0.66 h-l. Stationary phase began at about 12 h with a cell concentration of approximately 1.0 g/L. Gum production was both growth-associated and non-growth-associated, although the bulk of the gum was produced during the stationary (non-growth) phase. At 45 h, the gum concentration was approximately 26 g/L and the yield of gum on lactose was 0.66 g/g. The overall fermentor volumetric productivity for gum a t this time was approximately 0.6 g/(L.h). A maximum volumetric productivity of 0.8 g/ (Leh),was obtained a t 28.5 h and 23 g/L gum Concentration. The major byproducts of the fermentation were acetate, lactate, succinate, and carbon dioxide (Table 11). Trace amounts of 2,3-butanediol were detected near the end of the fermentation. The time course for the production of the organic acids is shown in Figure 2B. Some acids were produced in the early part of the fermentation during rapid cell growth and were then consumed by 24 h. A second phase of acid production occurred after 24 h. This may be a response to oxygen limitation due to the high gum concentration and broth viscosity at this time (Figure 3). Unfortunately, dissolved oxygen was not measured at this point due to a probe failure. As reported in the preceding paper (Flatt et al., 19921, Erwinia ATCC 55406 is a
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0
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Time, hr
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e0
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Time, hr Figure 3. Relationship between broth viscosity and lactan gum concentration during the fermentation of lactose by Erwinia sp. ATCC 55046 for the fermentation in Figure 2.
A
f
/ ' " " ' ' " l ' ' ~ ' I " ' ~ / ' " ' I
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€0
Time, hr Figure 2. Kinetics of fermentation of ladose by Erwinia sp. ATCC 55046 in a 5-L fermentor. (A) dry cell weight (O),polysac, lactose ( 0 ) . (B)Organic acid production: charide gum ( 0 ) and acetate (B), lactate (A),and succinate (0). Table 11. Product Concentrations and Yields a t 45 h for the Lactose Fermentation Shown in Figure 2 yield, g of product concn, g/L product/g of lactose 26.3 0.66 gum 2.3 0.06 acetate 1.8 0.05 lactate 0.9 0.02 succinate 2,3-butanediol 9 COz (gaseous) 0.31 0.9 0.02 cell mass 40.0 lactose consumed,g/L 106 % carbon recovered 0
Not detected until after 50 h.
facultative anaerobe. It is expected that gum concentration and productivity could be increased by improving oxygen trahsfer. Lactose Transport. In order to begin to understand why lactan gum production is better on disaccharidesthan on monosaccharides and to provide the foundation for future metabolic analysis and modeling, the mechanism of lactose transport in Erwinia sp. ATCC 55046 was investigated. The results of the study (Table 111)indicate that a permease system is the dominant mechanism of lactose transport in the organism. The turnover rates of ONPG, a lactose analog, were significantly higher than the turnover rates of ONPG-6-P, a lactose-6-P analog, in all three cell preparations. There was essentially no turnover of ONPG-6-P in permeabilized, toluene-treated cells or cell extracta, strongly indicating the absence of a phospho-8-galactosidase. Furthermore, the data indicate that permease transport is the rate-limitingstep for ONPG
Table 111. Turnover Rates of ONPG and ONPG-6-P in Various Cell Preparations of Erwinia sp. ATCC 88046. turnover rates rrmol/(ma DCWamin) cell preparation ONPG ONPG-6-P whole cells 0.13 3.8 X lo-' toluene-treated 0.17 ND permeabilized cells cell extract 0.38 ND The standard error of the measurements is approximately 25% of the indicated values on the basis of independent runs. Abbreviations: ND, not detected; DCW, dry cell weight; ONPG, o-nitrophenyl galactoside;ONPG-6-P,o-nitzophenyl&mgalactopyranoside 6-phosphate.
hydrolysis in the two-reaction system of permease and 8-galactosidase. The turnover rate for the whole cell preparation, which contains both permease and 8-galactosidase activities, was approximately one-third less than for the cell extract preparation with 8-galactosidase activity. This indicates that lactosetransport is potentially a rate-limiting step in gum production from lactose in Erwinia sp. ATCC 55046,assuming that the ONPG results can be extended to lactose. The mechanism of sugar transport may provide some insight concerning the difference in gum production on disaccharides versus monosaccharides. In facultative anaerobes such as the Erwinia, monosaccharides are generally transported by a phosphotransferase (PT) system and disaccharides are generally transported by a permease system (Saier, 1977). The PT system for glucose results in the generation of intracellular pyruvate. In the presence of high extracellular sugars (especiallyglucose), the pyruvate cannot be converted to sugars via gluconeogenesis (Moat and Foster, 1988)and must be metabolized to cell mass or organic acids. As shown in this paper, the glucose shake-flask fermentations generated more acids than the lactose fermentations. Since pyruvate generation is not coupled to permease transport systems, a larger fraction of the carbon in sugars transported permease systems is available for polysaccharide synthesis than for sugars transported by PT systems. Toprove that sugar transport is the reason for improved lactan gum production on disaccharides, well-controlled fermentationsmust be carried out with glucose and l a c h e . Experiments are also needed to determine the mechanism glucose transport in Erwinia sp. ATCC 55046. It should be noted that Erwina tahitica is reported to make the polysaccharide, Zanflo-10, more efficiently from sucrose and maltose than from glucose (Sandford, 1979). In conclusion, the research described in this paper and
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in the preceding paper (Flatt et al., 1992) provides the basis for the development of a new fermentation process for the production of a potentially useful gum from lactose in whey or whey permeate. It also provides insight into the utility and possible advantages of lactose as a fermentation substrate.
Acknowledgment We acknowledge Mr. Andreas Teske and Mr. Norm Voigt for assistance with the lactose transport experiments. We also acknowledge the input and support of Professor Norm Olson, Director, Center for Dairy Research, Madison, WI. This work was funded by the Wisconsin Milk Marketing Board and the State of Wisconsin, Economic Development Fund.
Literature Cited Flatt, J. H. Microbial Production of Novel Polysaccharides from Lactose in Whey Permeate. Ph.D. Dissertation, University of Wisconsin, Madison, WI, 1990. Flatt, J. H.; Hardin, R. S.;Gonzalez, J. M.; Dogger, D.; Lightfoot, E. N.; Cameron, D. C. An Anionic Galactomannan Polysaccharide Gum from a Lactose-Utilizing Bacterium. I. Strain
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Description and Gum Characterization. Biotechnol. Progr. 1992,preceding paper in this issue. Horton, B., Horton International,Inc., Cambridge,MA. Personal communication, 1992. Kornberg, H. L. Genetics in the Study of Carbohydrate Metabolism by Bacteria. J. Gen. Microbiol. 1976,96,1-16. McKay, L. L. Regulation of Lactose Metabolism in Dairy Streptococci. In Developments in Food Microbiology; Davies, R., Ed.; Applied Science: Essex, 1983;Vol. I, pp 153-182. Moat, A. G.; Foster, J. W. MicrobialPhysiology, 2nd ed.; J. Wiley & Sons: New York, 1988; pp 190-193. Pace, G. W. Microbial Gums. In Basic Biotechnology; Bu'lock, J., Kristiansen, B., Eds.; Academic Press: London, 1987;pp 449-462. Saier, M. H. Bacterial Phosphoenolpyruvate: Sugar Phosphotransferase Systems: Structural, Functional and Evolutionary Interrelationships. Bacteriol. Rev. 1977,41,856-871. Sandford, P. A. Exocellular Microbial Polysaccharides. Adv. Carbohydr. Chem. Biochem. 1979,36,265-313. Zabriskie, D. W.; Armiger, W. B.; Phillips, D. H.; Albano, P. A. Traders' Guide to Fermentation Media Formulation;Traders Protein: Memphis, TN, 1982;p 17. Accepted May 29, 1992. Registry No. Galactomannan, 11078-30-1;lactan, 14218813-4;lactose, 63-42-3.
