EFFECT ON FACTORY CANE JUICES AND SIRUPS OF L E U C O N O S T O C M E S E ~ ~ T E R I O I D E S ISOLATED FROM FROST DAMAGED

LOUISIANA SUGARCANE OF T H E 1937 CROP

M. A. MCCALIP, Carbohydrate Research Division, and

H . H. HALL, Food Research Division,

Bureau of Chemistry and Soils, U . S. Department of Agriculture, Washington, D. C. I

Difficulties are usually experienced in processing juice from cane that has been standing during a freeze. The rapidity with which operating difficulty ap- pears and the exact nature of the change in the juices are found to vary somewhat after each freeze. Usually when warm weather follows a freeze, the sugar factories are immediately confronted with rapidly increasing acidities accompanied by di- minishing trouble from "gumminess." At other times, usually when the freeze is followed by cool or cold weather, the muds become difficult to handle and crystal- lization of sirups is practically impossible with slightly more than normal acidity and with apparently well defecated juice.

This latter condition existed after the freeze which occurred in Louisiana in November, 1937. In most sections of the sugar belt, freezing temperatures were experienced on November 20, 21, and 22, registering a minimum of 26O F. in some sections. Subsequently, cold, dry weather prevailed for'the remainder of No- vember and the first few days of December. Then on Deceinber 6, 7, 9, 10, and 11, freezing temperatures were experienced again with cold weather following until December 13, when it turned warm. With four or ,five days of this warm weather the juice from the cane, much of which was cut and topped normally before trouble was anticipated, was in such condition as practically to stop factory opera- tion temporarily. After cleaning out this worst contamination of gummy juice and topping bacl; cane in the fields, conditions were relieved greatly and some of the low-topped standing cane was ground as late as the middle part of January.

I n addition to factory difficulty much trouble was experienced in the labora- tories in clarifying juices, sirups, and sugars which were contaminated with prod- ucts of fermentation. Usually much doubt must be placed on the correctness of the sucrose values obtained after analysis under these conditions.

This study was undertaken with the purpose of isolating and identifying the gum-producing organism and studying the conditions favorable to this viscous fermentation. I t was planned to produce the gum in culture media in the labora- tory in order to study the products of fermentation and their effect on sugar solu- tions. I t was hoped that a better knowledge of the properties of these products would be helpful in eliminating the difficulty they cause in the sugar factory and laboratory.

I Pasteur (1) was among the first to show that the slimy fermentation of carbo-

hydrate materials is due to bacterial action. Other early authors who worked with microorganisms producing slime in sugar solutions were Cienkowski (2) and Van Tiegham (3) . Cienkowski reported the isolation of a chain-forming coccus which produced large gelatinous capsules of slime when inoculated into sugar solutions. He gave the name Ascoccos mesenterioides to this strain with which he worked. The same year Van Tiegham worked with what appeared to be the same organism and gave it the generic name Leuconostoc.

Zettnow (4) isolated and studied in detail two types of organisms producing slime in sugar solutions to which he gave the generic name Streptococcus. The organisms described by Zettnow closely resembled those described by Van Tieg- ham. The results of Cienkowski, Van Tiegham, and Zettnow have formed the basis around which has been developed the knowledge of the Leuconostoc group of organisms.

More recently Hucker and Pederson (5) have contributed much valuable in- formation to the classification of the species of the genus Leuconostoc. Based on the total amount of acid produced in solutions of a variety of carbohydrates and polyhydric alcohols, they have defined the following three species: L. mesenter- ioides, L. dextricanus, and L. cilrovorus. Of the three named species, L. mesen- terioides is probably most often encountered in sugar solutions and by virtue of its ability to produce a slime or viscous fermentations is oi: most concern to the

Scheibler [Lafar (6)], isolated a mucilaginous material from the juice of sugar beets which he identified as being an anhydride of glucose, closely related to starch and dextrin, to which he gave the name "dextran." Variations in the pro- duction of dextran have been found to exist among various species of Leuco- nostoc; however, it has been determined that sucrose is an essential ingredient of the medium for its production. Tarr and Hibbart (7) observed that slime forma- tion from glucose is a somewhat transient factor, occurring only when the cultures are very active in respect to the formation of dextran from sucrose.

Browne (8) was among the first workers to demonstrate the action of bacteria in frozen sugarcane. He showed that the natural antiseptic property of cane stalks was lost upon freezing and that the hordes of bacteria entering the split stalks start a fermentation which renders the cane worthless for milling. Owen (9) demonstrated that the temperature at which cane juice was fermented con- trolled the type of fermentation. Juices that were held at temperatures of from 50-550 F. were more susceptible to the viscous or gummy type fermentation, whereas juice held at temperatures twenty degrees higher were more apt to un- dergo alcoholic and acetous fermentation. More recently, Owen (10) discussed the bacteriological aspects of frozen cane from the standpoint of control measures that may be utilized to prevent losses.

ISOLATION AND IDENTIFICATION OF L. mesenterioides

The cultures of the gum-forming organisms were isolated from stalks of frost-damaged cane which had been shipped from Louisiana to the Washington laboratory. Stalks of cane which had been windrowed after the first and second freezes and those standing after the second freeze, all showing evidence of a

987 I

"splitting freeze," were used. Several stalks of cane representing each of the above conditions of handling were divided into sections; each section containing two nodes and a complete internode which was equally divided about the nodes was peeled and ground in a food chopper. Ten grams of each section was weighed into 90 grams of sterile water for dilution plating. The plates were poured with a culture medium composed of 12 per cent gelatin and 5 per cent sucrose which was adjusted to pH 6.8-7.0.

The plates were incubated at 20° C. for seven days before attempting to pick representative colonies. After 48 hours of incubation small white to greyish col- onies appeared arid usually after four days the maximum number of colonies had developed. Maximum colony size and characteristics were obtained in seven days. The col'onies measure from 2 to 8 mm. in diameter, and are raised, glistening, and frequently rough and viscous. Characteristic colonies were picked and trans- ferred to gelatin-sucrose medium for further study.

I t was observed that the heaviest infection of gum-forming bacteria was most often in the upper three joints of a stalk regardless of its soundness. Heavy in- fection in lower joints was usually associated with damage resulting from borer infestation and splits in the stalk which facilitated access and subsequent growth of the organisms.

The cells of typical gum forming cultures are gram positive, non-motile, spheres measuring from 0.5 to 1.0 microns in diameter, occurring singly, in pairs, and in short chains. Acid is produced from sucrose and the pentoses, xylose and arabinose. Slime is produced from sucrose, its production being enhanced by the addition of yeast extract to the medium. There is no production of slime from glucose. The optimum growth temperature is 20-22O C. The oxygen requirements are facultative.

Since the morphological, physiological, and cultural characteristics of the cul- tures correspond in all the essentials to Bergey's (11) description of Leuconostoc mesenterioides (Cienkowski, Van Tiegham) , the predominating gum-forming or- ganisms are considered to be members of this species.

I GROWTH OF L. mesenterioides IN SUCROSE SOLUTIONS

In, order to translate in terms of the effect on solution properties of the growth of the organisms on sucrose, it seemed desirable to employ a medium con- taining only sucrose. This was proved impossible, however, and it was found ne- cessary to provide an accessory food substance for the cultures. The addition oi powdered yeast extract was found sufficient to promote rapid growth of the cul- ,

tures in sucrose solutions with the subsequent production of viscous fermentation. Therefore, the studies were made by growing pure cultures of the organisms in 10 per cent sucrose solutions which contained 0.20 per cent Bacto yeast extract. The reaction of the solutions after sterilization was slightly alkaline.

In an experiment 500 cc. of sucrose solution was placed in each of ten one- liter flasks, plugged with cotton and sterilized at 15 pounds pressure for 20 min- utes. When cooled one cc. of a water suspension of the culture was pipetted into each flask of solution. The flasks were incubated at 20° C. for various periods of time and were then removed for the following determinations: Solids (by refrac-

tometer), direct sucrose, invert reading, true sucrose by invertase, mgs. of invert sugar per 100 grams, pH, titrated acidity, mgs. of crude gum precipitated from 10 cc. of solution with five volumes of 95 per cent alcohol, viscosity by Englen's method (12), and the number of bacteria per cc. of solution determined on su- crose-gelatin medium. The results of the examination of the samples from such a series are given in table 1.

A rapid increase occurs in the number of bacteria immediately following inoculation of the samples and this is accompanied by changes in solution proper- ties. Most noteworthy of the changes are increased acidity, crude gum content, and viscosity. Following the maximum development of the culture, which occurs in about 24 hours, there is a rapid reduction in the plate count which is probably due to the formation of bacterial by-products, notably acid. It is evident that bac- terial metabolism is not inhibited after 24 hours because paralleling this reduction are further increases in acidity, crude gum, viscosity, and invert sugar and de- creased sucrose content.

However, it was noted in this series and in other cultural solutions that the viscosity increase and the destruction of sucrose was practically arrested in lightly buffered media when enough acid is produced to drop the solution to around pH 4.0. Unless this acid is neutralized any further action that goes on tends grad- ually to reduce the viscosity.

One solution which contained 21y0 sucrose and 0.2% Bacto yeast extract when inoculated showed the following analysis after 48 hours: sucrose 18.5%, re- ducing sugars 1.08%, crude gum 1.3%, viscosity (Engler) 1.58, and pH=4.8. After allowing the fermentation to continue for 10 days more, the viscosity had increased to only 1.702, crude gum content to 1.8y0, and the pH dropped to 3.96. At this stage a buffer solution containing sodium phosphate and potassium chlor- ide was added and the pH adjusted to 7.5 with 2y0 NaOH and the viscosity de- termined at intervals during the next 20 days with the following results:

Days after buffer and neutralization Viscosity

of acid (Engler)

2 days 4 "

7 "

19 "

After precipitating the dextran from this solution, the alcoholic filtrate was found to .have a negative polarization. Acid hydrolysis showed, however, there still remained approximately 1.37% sucrose in solution.

I n order to obtain higher concentrations of the products of fermentation for purification and study, the pure cultures were grown in 10 and 20y0 sucrose solu- tions that were buffered at a pH of 7.5 to start and the acid neutralized at inter- vals of 48 hours with 2% sodium hydroxide under aseptic conditions. Under these conditions the fermentation continued until the culture medium was transformed into a thick, gelatinous mass of high viscosity, the same as has been encountered with cane juice by Browne (8) and other investigators.

At the end of 24 hours after inoculation the culture medium becomes turbid and viscous with a white sediment settling at the bottom of the flask. Within a few days the gelatinous mass takes on an opalescent appearance. When a sugar solution has undergone this fermentation to only a slight degree, it becomes very difficult to filter, even using large quantities of filter aid, unless the gummy sub- stance formed is first precipitated. Without first eliminating the gum the filtrate must be filtered two or three more times to eliminate the turbidity and then the solution is opalescent. Usually a small amount of ammonia or dilute alkali will render the solution transparent. It was observed that when using filter aid for filtering solutions of this gummy substance particles of filter aid appeared to be- come entangled with the gum in the colloidal phase and to remove part of it. Repeated filtration with filter aid disclosed that the polarization of the solution was lowered each time the solution was filtered.

SEPARATION AND PURIFICATION OF BY-PRODUCTS

The buffered solution after undergoing fermentation was treated with five volumes of 95y0 alcohol to precipitate the gum. The gum first appeared to come out in stringy white flocks which soon stick together forming a mass which is dif- ficult to wash free of sugars and other impurities. This crude gum after kneading -, and washing with alcohol was repeatedly purified by dissolving in water with the addition of a small amount of dilute sodium hydroxide and precipitating with alcohol acidified with acetic acid. In several cases the crude gum when put into solution the first time showed a specific rotation of only 60 to 65 and an ash con- tent of around 2.0y0. With repeated purification the specific rotation could be in- creased to + 195 with five or six precipitations, after which further treatment did not increase the specific rotation consistently. The ash of the purified gum was found to be 0.2070 to 0.40y0. The purified gum solution showed no reducing ac- tion when tested with Fellling solution.

A solution of the purified gum containing 1.5806 grams solids per 100 cc. PO-

larized 8.90° V. in a 100 mm. tube at 20° C., which corresponds to a specific rota- tion of 195.150. After hydrolysis with hydrochloric acid, the polarization of a soh- tion of equivalent concentration was found to be f 2.54O. Reducing sugar was determined on the hydrolyzed solution. Calculated as dextrose, a concentration of 1.712 grams per 100 cc. was found. Calculating the specific rotation from the concentration of 1.712 and polarization of +2.54 gave a specific rotation of 51.42O for the reducing sugar produced by hydrolysis. The results indicated that dextrose only, which had a specific rotation of 52.5, was produced when this gum was

The general properties of the gum produced by the fermentation in this study appear to coincide with the product named dextran by Scheibler (6) and usually

' referred to by other authors by the same name. Some authors give the specific rotation for dextran as +ZOO and it is possible that some impurities still remained in these samples.

The dextran studied in this case did not appear to be entirely white in the hydrated form after purification. There was a slight yellowish cast that was more in evidence in the pure than the unpurified product. After drying and pulveriz-

ing it is a nonhygroscopic white powder. Dextran appears to become less soluble in water as purification progresses.

CRYSTALLIZATION OF D-MANNITOL

The culture medium after removal of the dextran with alcohol was evap- orated to a sirup of 72 per cent solids and allowed to stand at room temperature overnight. Very fine needle-like crystals crystallized out causing the mass to set up solid. This mass was diluted slightly by adding a little distilled water and stir- ring. After warming to 60° C. the crystals were filtered off on a Buchner funnel using vacuum and washed with 60% alcohol. The mother liquor was concentrated on the steam bath at low temperature and allowed to set overnight when a second crop of crystals was obtained.

The crystalline material was purified by dissolving in water and recrystalliz- ing two or three times using decolorizing carbon when necessary. The material was found to crystallize out of water solution of 40y0 solids immediately upon cooling, forming a mass of fine needle crystals. Strong alcohol precipitates manni- to1 from rather low density water solutions.

These crystals after drying at low temperature in a vacuum oven had a melt- ing point of 1660 C.; a concentration of 6.8850 gms. to 100 cc. polarized -0.08O in 100 mm. tube at 20° C. This polarization corresponds to a specific rotation of -0.40°. These crystals were also polarized in borax solution, giving a specific rotation of 24.68O at 20° C. The above properties appeared to identify the mate- rial as d-mannitol.

Using the method given by Walton and Fort (13) in their previous study along this same line, the tribenzol derivative of the d-mannitol was prepared. It consisted of a white felty mass insoluble in water and soluble in toluene which showed a melting point after purification of 220° C., which is the melting point given in the literature for tribenzol mannitol. The optical properties of both the d-mannitol and' tribenzol mannitol were tested by Keenan" and found to be identical with measurements previously obtained for these products.

All of the cultural solutions when tested for mannitol polarimetrically using the arsenic trioxide method of Badreau (14) showed the presence of mannitol, but crystallization was not possible from such solutions until fermentation had de- stroyed most of the sucrose present.

The residue, after removal of dextran and mannitol, was found to consist principally of reducing sugars. Of the reducing sugars present determination of dextrose by iodine oxidation using the method of Lothrop and Holmes (15) and calculating levulose by the difference between total reducing sugars and dextrose showed dextrose to predominate in all solutions tested. The average of three de- termination3 showed dextrose as 76.92 per cent of the total reducing sugars and levulose as 23.12y0.

A STUDY OF THE ACTION OF CLARIFYING AGENTS FOR LABORATORY ANALYSIS

Normally, most sugar factory laboratories use Horne's dry lead subacetate method for clarifying juices, sirups, and other sugar-house products for sucrose

# G . L. Keenan, Microanalyst, Food and Drug Administration.

analysis. When these products have partly undergone the viscous fermentation, clarification and filtration becomes a problem. The dry lead method becomes very ineffective and the normal weight method using lead subacetate solution has to be employed. If the juices are badly fermented, the same difficulty of poor clarity and slow filtration rate is experienced with all the products from juice to sugar. In order to analyze these products in the time available, it is necess&ry to use lead subacetate solution together with about 50 cc. of alcohol per normal weight. Under these conditions the sucrose figure obtained must be questioned.

In order to study the results obtained, juice was expressed from one sample of sound cane that was windrowed before the freeze and two samples of standing cane that was not windrowed. Sucrose was determined using Horne's method with dry lead subacetate, lead subacetate solution added to normal weight, and lead subacetate solution plus alcohol added to normal weight. The results are tabulated in table 2.

I t can be seen from the table that the direct sucrose, which is of most impor- tance to the sugar factory, and also the true sucrose check reasonably well where dry lead subacetate aqd lead subacetate solution only are used on sound cane. The high direct and low invert polarizations where alcohol was used are explained by the fact that alcohol reduces the specific rotation of levulose greatly. In both analyses of the deteriorated juice the direct sucrose is shown to be much higher when using Horne's method than when the normal weight method is used and in every case higher even that the indicated true sucrose. I t is of interest to note the low invert polarization as compared to the direct polarization, especially when the solution was clarified with dry lead subacetate.

A study of the laboratory clarification of juices and cultural sugar solutions that have undergone the viscous fermentations leads to the conclusion that the excess acidity, together with the very large amount of lead subacetate required to precipitate dextran completely, is responsible for the ineffectiveness of this re- agent. Attempts to clarify such solutions with either dry lead subacetate or lead subacetate solution result in much of the lead going into solution before even slow filtration is possible. The dry lead subacetate method is least effective because of the greater effect of excess acid and the larger quantity of dextran that must be precipitated from the larger quantity of sample used. The quantity of lead subacetate, either dry or in solution, necessary to effect clarificatiorl is much greater than would be expected since the addition of one to two per cent of dex- tran on solids just about doubles the lead requirements.

Using the normal weight method and clarifying with lead subacetate solution, it was found that even very badly deteriorated solutions would clarify if the cor- rect amount of lead was added within very narrow limits. It was noted that the quantity required was teduced by adding a few drops of dilute ammonium hy- droxide or other dilute alkali to raise the pH to 6.5 to 6.7, though addition of sufficient alkali to change the reaction of the filtrate to alkaline must be avoided. The difficulty of determining the quantity of lead subacetate solution within nar- row limits, the amount of lead going into solution, and possibly the greater quan- tity of reducing sugars precipitated by the large quantity of lead subacetate required makes this method far from satisfactory.

993

TABLE 2.

COMPARISON OF RESULTS WHEN ANALYZING JUICE FROM SOUND AND DETERIORATED CANE USING HORNE'S METHOD AND NORMAL WEIGHT METHOD CLARIFYING WITH AND WITHOUT THE USE OF ALCOHOL.

Acidity cc. Method of Direct Acid Invertase True True Material N/100 NaOH Method Clarify- Polari- Invert Invert Direct Sucrose Sucrose

per 10 cc. ing zation Reading Reading Sucrose Acid Invertase

Juice from windrowed cane. . . . . . 2.6 Horne'sMethod Dry Lead 52.68 -20.34 -19.94 12.86 13.42 13.44 '1 11 u U . . . . . 2.6 N/Wt. L.S.A. 12.76 -4.84 -4.76 12.76 13.31 13.34

Sol. 11 LL 11 n ...... 2.6 N/Wt. L.S.A. 13.10 -4.30 *-4.04 13.10 13.15 . . . . .

Sol. +Ale. Juice from deteriorated cane.. . . . 8.7 Home'sMethod Dry Lead 35.35 -3.22 -1.80 8.71 7.22 6.97

u 11 11 n 8.7 N/Wt. L.S.A. 7.25 -2.00 *-2.34 7.25 6.98 7.28 Sol. +Ale.

Juice from deteriorated cane. . . . . 7.7 Horne'sMethod Dry Lead 42.5 -6.64 -5.50 10.37 9.02 8.87 U 11 u il ..,.. 7.7 N/Wt. L.S.A 9.58 ..... -2.04 9.58 ..... 8.85

Sol. u 41 U " ..... 7.7 N/Wt. L.S.A. 9.42 -2.70 *-2.50 9.42 9.17 9.08

* Alcohol removed before invertase added.

I

A number of clarifying agents were tested with this type of solution in search of a reagent that would give better clarification and filtration than lead subace- tate. I t was found that Herle's basic lead nitrate method was much more effective in precipitating the gum from this type of solution than the more commonly used methods.

This method involves the use of two solutions. Solution (1) is prepared by dissolving 1,000 grams of neutral lead nitrate in 2,000 cc. of water and solution (2) is prepared by dissolving 100 grams of pure sodium hydroxide in 2,000 cc. of water. The clarification is most effectively carried out by adding a measured quan- tity of the lead nitrate solution (2 cc. to 15 cc. according to material to be clarified) first and mixing well and then adding an equal quantity of the second solution which precipitates basic lead nitrate in the solution to be clarified. After prepara- tion, the solutions should be checked against each other and adjusted to give an acid reaction very close to neutral.

This method was tested on various types of solutions containing dextran and other products of fermentation and found to eliminate the dextran almost quan- titatively with a brilliant filtrate and good rate of filtration when used properly. Excess acidity increased the quantity of these solutions required for effective clari- fication and also the amount of lead going into solution, but to a less extent than when lead subacetate was used, Also, it offered the advantage of easily counter- acting high acidity by slightly increasing the quantity of sodium hydroxide, al- though addition of an excess which would produce an alkaline reaction must be avoided.

Table 3 shows the effect of clarifying various sucrose and dextran mixtures, using Herle's basic lead nitrate method. In all cases the clarity was good and the rate of filtration rapid.

The method was further tested using a table sirup diluted to 56.5y0 solids (by refractometer) and containing a high percentage of reducing sugars to deter- mine the extent to which reducing sugars are precipitated. Comparison was made by determining sucrose by use of the method of Balch* which was found to pre- cipitate no reducing sugars. This clarification involves the use of equal quantities of a saturated solution of neutral lead acetate and a 6% tannic acid solution. The results are tabulated in table 4. When using a solution of this type with high reducing sugar content, it is noted that sufficient reducing sugar is precipitated to increase the apparent purity by about one per cent when using the basic lead nitrate method as compared with 1.2% when basic lead acetate is used. This ex- periment confirms results obtained by Bryan (16) in his study of various clari- fying agents in 1906.

Two per cent dextran on solids was added to the same sirup and diluted to 48.6 solids and the sucrose and apparent purity were determined using basic lead nitrate and lead subacetate solutions only, as it was not possible to clarify a solu- tion containing this quantity of dextran with neutral lead acetate and tannic acid. Two or three determinations were made with each clarifying agent using different quantities of reagent to clarify. The results are tabulated in table 5.

I * R. T. Balch, Carbohydrate Research Division.

TABLE 4. TABLE SIRUP CONTAINING HIGH INVERT SUGAR CONTENT CLARIFIED WITH DIFFERENT CLARIFYING AGENTS -

Conc. Amount of Ref. Clarifying Agent Per Clar. Solids Sucrose Purity

100 cc. Agent cc.

Equal Quantities of Saturated Neutral Lead Acetate and 6% Tannic Acid ............. .......................................... Solutions. : 26 gms. 4 cc. ea.

Herle's Basic Lead Nitrate Method. ................................... 26 gms. 5 cc. ea. 56.50 27.71 49.04

Lead Subacetate Solution. ............................................ 26 gms. 8 cc. ea. 56.50 27.86 49.31

TABLE 5. ANALYSIS OF TABLE SIRUP USED IN TABLE 4 DILUTED AT TIME OF ADDING 2 % DEXTRAN ON SOLIDS.

Conc. Clarifying Refractom- Clarifying Agent per Agent Used eter Sucrose Purity Remark

100 cc. CC. Solids

..... Herle's Basic Lead Nitrate Method. NjWt. 8 cc. ea. 48.6 24.85 N/Wt. 10 CC. ea. 48.6 25. 08 51.60 Good clarity and filtration rate.

Lead Subacetate Solution. N/Wt. 16 cc. 48.6 24.85 51.13 Filtered very slowly.

i . . . . . . . . . . . . .

+ NXOH

NjWt. 18 cc. 48.6 25.20 51.85 Filtration rate fair.

NjWt. 22 CC. 48.6 25.80 53.08 i t LC u

The resulting purity obtained for the sirup was found to be increased over that found in the preceding table by about 3y0, using the absolute minimum of reagents. The use of only slightly more clarifying agents in each case raises the purity figure. This increase of purity is attributed in both cases to precipitation of reducing sugars, of which approximately 30% on solids are present, and not to dextran left in solution. Testing the deleaded filtrate with alcohol for dextran substantiated this conclusion. When sugars and products containing a small per- centage of reducing sugars are clarified, the error due to this cause would not be pronounced.

THE EFFECT OF DEXTRAN AND OTHER PRODUCTS OF FERMENTATION ON THE VISCOSITY OF SUCROSE SOLUTIONS

Since the effect of the fermentation products on viscosity was so evident, a precise method of measuring viscosity at various known concentrations was em- ployed. The Bingham (17) and Greene type of viscosimeter was used for this in- vestigation. This instrument has an advantage over the Ostwald type in eliminat- ing the error which may result from a slight change in the working volume, but depends upon air presspre rather than gravity to force the liquid through the capillary. Viscosimeters were available with different size capillaries which had previously been calibrated with oil of known viscosity to determine the constant. Two viscosimeters were used which had constants of .3956x10-6 and .6165~10-~. These viscosimeters were attached to frames which held them rigidly in place in a vertical position in the water bath.

The water bath used to insure constant temperature of the solutions while passing through the capillary was especially constructed for this type of viscosi- meter and had metal sides and bottom with a glass front and back, in order that the flow of liquid in the viscosimeter could be observed when it was immersed in the bath. The bath was equipped with a well type, motor driven stirrer for cir- culation and heated by means of electric heaters. A mercury temperature regula- tor of customary type in conjunction with a relay gave a regulation in temperature of &0.020 C . A Beekman thermometer was used for making these observations.

T o obtain a source of air pressure maintained constant over long periods of time, the Dawson (18) precise pressure regulator was used. This regulator makes possible the regulation of air pressure in a reservoir by allowing excess air to escape automatically through specially constructed regulating tubes. This regula- tor maintains a constant pressure with a variation of -10.1 mm. It was fitted with a telescopic reading device mounted on a frame and actuated through pinions and racks and calibrated to read the distance between the levels of mercury in the two arms of a U tube having one end connected to the reservoir and the other end open to the atmosphere. Temperature observations were made on the man- nometer board, in order to correct the pressure for varying temperatures.

A solution of dextran of high purity was prepared with known solid content for use in making up sucrose-dextran mixtures. The solution of fermentation products and crude dextran used in the determinations was prepared by filtering several times through a thickness of three filter papers on a Buohner funnel under vacuum to obtain a solutiop free from suspended matter. ~ol'ids determinations

were made on these solutions at 710 C. in vacuum oven. Solutidns for determina- tions were weighed out in the proportions desired at approximately 66y0 solids and the actual soiids content determined in triplicate. Since it was desired to work at a density below the saturation point, and in view of the availability of data on viscosity of sucrose solutions at 400 C. obtained by Hill*, it was decided to make these observations at 65.23% solids and 40° C. '

In order to guard against interference of dust particles, air bubbles and water evaporation, the following procedure was adopted for preparation of the 20-gram samples of solution for the viscosimeters. Exact quantities of distilled water and solutions of known solids content were weighed into a weighing bottle. The bottle was closed and the solution mixed thoroughly and centrifuged for 20 minutes be- fore transferring to the viscosimeter, using a special pipette with dry, dust-free air to force in the correct amount. The transfer was not made until temperature and pressure were checked and found to be under exact control. After volume ad- justment, the observations of the time required to pass through the capillary tube were made in both directions and the mean was used for calculation of viscosity in poises. In many cases viscosity was determined at both high and low pressures in order to detect existence of a plastic effect,

Viscosity was first determined on a pure sucrose solution of 65.23y0 solids and was found to be 0.4620 poise which checked closely with results obtained by Hill*, and by Bingham and Jackson (19). Observations were next made using 3.36% of pure and of crude dextran on solids; also 3.36% crude dextran plus 3.79% of fermentation products solids replacing that quantity of sucrose. The re- sults are tabulated in table 6.

The conclusion to be drawn from the viscosity data is that the pure dextran has a much greater effect on viscosity than the same percentage of crude dextran or an equal percentage of crude dextran plus a more or less equal amount of invert sugar, mannitol and other solids present in the solution after viscous fer- mentation. The viscosity produced by 3.36% of pure dextran on solids is more than double that found when using an equal quantity of crude dextran. The dif- ference in viscosities produced by 3.36% crude dextran and 3.35% crude dextran f 3.79% of other solids is very slight, the latter being a little higher. This indi- cates that the increase in viscosity after fermentation is due primarily to dextran rather than to other products formed during fermentation.

Determination of viscosity of sucrose solutions plus various concentrations of pure dextran ranging between 0.0 and 6.0% were made at approximately 283 grams per cmZ and 545 grams per cm2 pressure. The results are tabulated in table 7.

The viscosities obtained are found to be higher at the lower than at the higher pressures, in every case with the differences much greater at the higher viscosities, showing that there is a considerable plastic eaect in solutions of this nature. A curve was plotted using the results recorded in table 7. From the curve it is shown that addition of 1% of dextran on solids a little more than doubles

+ H. G. Hill, formerly of the Carbohydrate Research Division.

Viscosity in , Viscosity in Sample Total Solids Sucrose Dextran Poise at Poise at Number of % % 283 gms. per 545 gms. per

Solulion Solids Solids (Cm)2 Pressure (Cm)2 Pressure

the viscosity of a 65.23y0 sucrose solution at 400 C., and that addition of 6.01% dextran replacing the same quantity of sucrose increases the viscosity 37 times. The presence of 1.8% dextran, which is possibly somewhat above the quantity OC-

curring in sugar factories, increases the viscosity 3.9 times.

I t can be seen from the effect of increasing amounts of dextran why crystalli- zation of sucrose becomes so difficult when cane juice has undergone fermentation of this type. Since it is sometimes stated that clarification at a lower or higher pH improves the "workability" of this type of juice, further determinations were made to ascertain the effect of pH on the viscosity of mixtures of sucrose and dex- tran. The viscosity of two solutions of sucrose plus pure dextran of different per- centages and one solution containing sucrose and crude gum plus other fermen- tation solids was determined. The results are tabulated in table 8.

Viscosity in Viscosily in Sample Sucrose Dextran pH Poise at 283 Poise at 545 Number % % of gms. per Cm2 gms, per CmZ

Solids Solids Solution Pressure Pressure

GUM % TOTAL SOLIDS Viscosity of sucrose solutio~ls with different concentrations of dextran

at 65.23% solids at 40' C.

The data indicate that the pH of the solution has practically no effect on the viscosity, once the dextran is in solution. The viscosity values found are in some cases a little lower at the lower pH values, but are considered on the whole to be within experimental error.

A sample of effect sirup was obtained from a Louisiana sugar factory at the time presence of dextran was causing difficulty. After standing it jellied, as is often the case with badly fermented sirup, and had the appearance of containing around 80% solids. Analysis showed 69.5 solids and 68.4 purity. A sample for vis- cosity determination was prepared by diluting to 10% solids and filtering through a filter paper lightly coated with filter aid on a Biichner funnel under vacuum to remove suspended matter. The filtrate was concentrated to 66% solids under vacuum. The second sample was prepared by precipitating dextran with 4 vol- umes of alcohol, filtering and concentrating under vacuum and boiling at atmos-

pheric pressure for 10 minutes to remove alcohol. Viscosities were determined at 65.23y0 solids at 40° C. on both samples with the following results:

Sample Viscosity in Poises Gum-free Sirup . . . . . . . . . . . . . . . . . . . . . . 0.3824 Gummy Sirup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.5553

It is shown that the gummy sample has a viscosity a little more than 4 times that of the sample containing no gum. It is noted that the viscosity of the gum- free sample is lower than that found for pure sucrose. The possibility of some al- cohol remaining in this sample must necessarily cast some doubt on its correct- ness. However, it has been shown by E. W. Greet (20) and also Spengler and Landt (21) that many salts found in sugar-house products have a less effect than sucrose in increasing viscosity. From the percentage weight of gum precipitate and the specific rotation is was estimated roughly that this original sirup contained ap- proximately 2.0y0 pure dextran on solids.

Since cane juices were not available at this time for studying the question of dextran removal by clarification, synthetic solutions prepared from diluted cane syrup and dextran solutions together with dextran solutions prepared by cultur- ing the causative microorganism were used for this brief investigation.

Synthetic juices were treated with lime alone, sulphur dioxide and lime, and phosphoric acid and lime at various pH values. A portion of the resulting clarified juices was subsequently treated with 5 volumes of alcohol for precipitation of re- maining dextran present. After washing with alcohol, the precipitate was redis- solved, using a few drops of alkali, and the polarization of the solution was deter- mined to obtain a rough check on the elimination of the dextran originally pres- ent. The effect on elimination of dextran of superheating the juice before and after liming was also tested.

The use of these more commonly used processes under varying conditions did not prove to be very effective, the best elimination being only about 25y0 of the dextran present. This result was obtained by increasing the initial acidity 2 or 3 cc. of N/10 NaOH per 10 cc. with sulphur dioxide or phosphoric acid before liming to give a clarified juice of about pH 6.8. The advantage of this procedure appeared to be that the heavy flocculation and greater volume of mud entangled more dextran than a lighter flocculation. The type oE flocculation obtained by addition of phosphoric acid proved to be a little more effective than when SO, was used. Superheating to 240° F. before and after sulphuring and liming did not affect the elimination appreciably. Since dextran has so far been found to be precipitated only by the salts of the heavy metals and by dehydration, the problem of removal in clarification appears to be a difficult one.

SUMMARY

1. The fermentation responsible for the slow filtering muds and poor crystal- lizing sirups and molasses during the latter part of the Louisiana sugar season of 1937 resulted from viscous fermentation by the microorganism L. mesenterioides producing dextran.

2. This microorganism, probably entering the stalk through the frost-killed eye passages, borer holes and freeze splits, acts rapidly on cane of low germicidal resistance so long as pH and temperature are favorable.

3. The gum dextran was produced by the viscous fermentation of sucrose solutions. Crystals of d-mannitol were recovered from dextran-free solution resi- dues.

4. Sucrose determination in laboratory samples of solutions containing dex- tran is facilitated by clarification by use of ~Herle's basic lead nitrate method.

5. Of the products of viscous fermentation dextran is primarily responsible f ~ r increasing the viscosity of sirups and retarding filtration and crystallization.

6. Purified dextran, when added to sucrose solutions, materially increases the viscosity. One per cent of dextran on solids more than doubles solution vis- cosity, whereas 6 per cent dextran on solids was found to increase solution vis- cosity 37 times.

7. Only approximately 25 per cent of dextran in solution was removed by ordinary factory methods of juice clarification.

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