Development of during Sugar SIGNIFICANCE RELATIVE TO SUCROSE LOSSES Undetermined sucrose losses during the processing of cane juices for the production of sugar, or in the processing of raw sugar in refining, have not infrequently been attributed to the activities of microorganisms. Investigations of losses at high temperatures during raw sugar manufacture have shown that in the presence of either activated decolorizing carbon or refiner's boneblack losses of sucrose at 10' and 65' Brix may be very rapid. I t is assumed that these materials accelerate the normal activities of thermophilic bacteria, both in protecting them from high temperature and furnishing ideal conditions for anaerobic fermentations. It is probable that both juices and sirups are at times allowed to remain at temperature levels of approximately 132' F. for sufficient lengths of time to result in considerable losses in sucrose, due to microbial activities. The investigation is being continued under conditions more closely approximating plant operations, in the hope of more completely exploring the extent of this potential source of sucrose losses in refining.
WM. L. OWEN P.O. Box 1345, Baton Rouge, La.
N
OTWITHSTANDING the fact that knowledge of the microbiological activities in cane and beet sugar products has increased immeasurably during the past few decades, there is still lacking a definite evaluation of the possible role of micro0rganisms:as causative agents in the destruction of sucrose during sugar manufacture or in cane sugar refining. It has been almost three quarters of a century since the nature of the formation of dextran in extracted cane and beet juices and dilute sugar solution was discovered by Cienkowslu ( 5 ) and Van Tiegham ( 1 7 ) , and yet the identity of all the species of bacteria developing in cane juices and their power in destroying sucrose in mesophilic and thermophilic temperatures have not yet been appraised. For the past two decades the interest in the microbiology of the sugar industry has been almost entirely confined to the study of the sources and the occurrence of thermophilic bacteria in refined sugars. Since Cameron and Williams (3),Cameron and Bigelow (a), and others discovered that even the highest grade of refined sugars could be carriers of thermophilic species of bacteria which were highly detrimental to canned nonacid vegetables, the interest of the sugar refiner in sugar microbiology has been in the production of refined sugar of the highest purity, as regards its chemical composition and microbiological content. This interest has in recent years almost overshadowed that of the role of thermophilic microorganisms in the possible losses of sucrose during manufacture or refining. Proof of the fact that bot'h thermophilic and mesophilic microorganisms play a very significant part in sucrose losses in cane juices and light, liquors was provided a t least 30 years ago b y Carpenter and Bomonti (4), McAllep and Walker (IO),Owen ( l a ) ,and others. The experiments conducted a t the Hawaiian Experiment Station three decades ago showed, by indirect methods of appraisal at least, that the deberioration of cane juices during milling could be greatly reduced by frequent disinfection of the mill with a suitable germicide. It was shown that the losses in sucrose in cane juices during milling were highest a t the end of the week 606
before the washing down a t the mill and lowest immediately after this procedure. In recent years chlorine has been used as a disinfectant in mills, and under the Daniel sterilization process ( 7 ) it was used on the crushed and mixed juices as a preservative against the action of microorganisms. The inventors of this process have claimed that there was a gain of 0.54 in purity of the mixed juices, as a result of chlorination, which represented an increase in sugar recovery of 31,250 pounds for each million arrobas of cane ground.
SUCROSE LOSSES DUE TO MICROORGANISMS The ordinary losses of sucrose during milling, as a result of the activity of microorganisms, are normally very low. The fact that they normally occur is generally recognized and appreciated by mill operators The difficulty has been in accurately appraising these sniall losses. For example, a loss of 0.1% in polarization may be within experimental error, but if it occurred consistently it would represent a very large loss in the aggregate, when applied to the volume of juice in mills grinding more than 1000 tons per day. The possibilities of losses occurring in juices in transit from the mills to the juice heaters to the clarifiers, as a result of thermophilic bacteria, may be suspected from the experiments conducted a t the Hawaiian Experiment Station two decades ago by McCleery and others ( 1 1 ) . These authors found definite losses in purity in juices held for any length of time a t high temperatures and concluded that cane juice can more safely be held at a temperature above 160" and below 180" F. than in ranges within which thermophilic and mesophilic bacteria are active. I n some cases they found losses as high as 3% in purity in juices which had been held for 18 hours a t 80" C. More recently, De Whalley and Scarr (6) have referred to bacterial contamination in bone char filters of refineries which reinfected the liquor, although the char had been regenerated a t 500" to 600" F. It is more and more apparent that undetermined losses in sugar factory operations are becoming more serious and that too
March 1951
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INDUSTRIAL A N D ENGINEERING CHEMISJRY
little attention has been paid to the possible sucrose destruction by hitherto undetermined species of microorganisms. The application of microbiology to the problem of sugar manufacture has, up t o this time, been almost entirely confined to the prevention of microbial deterioration of cane juices or beet diffusion juices and to the production of refined sugars which meet the exacting demands of the canning trade, with reference to their freedom from the spores of thermophilic bacteria which are capable of inducing spoilage in nonacid type vegetables, especially corn. The methods used in the detection of viable spores of thermcphilic bacteria in sugars have been entirely unrevealing as an appraisal of the significance of these organisms as causative agencies in the inversion of sucrose a t high temperatures, during manufacturing operations. The reason for this is that the substrates used for the detection of these species of thermophilic bacteria, which are of concern to the canning trade using sugar in its products, are not designed t o reveal the role they might play as contributory agents in sugar losses, either during the manufacture of raw sugar or in its relining. The presence of these species in refined sugars is as a rule just as unrelated t o the inherent composition of the product as an appraisal of dust or other foreign particles might be. They may be regarded as entirely inert and of no significance whatever a t almost any concentration of the dilution of the sugar itself, as they are inhibited from growth not only by the temperature and lack of moisture in the product, but to a large extent also by the lack of suitable nutrient materials in clarified juice and dilute sugar liquors. On the other hand, unquestionably many hitherto unidentified species of thermophilic microorganisms are not only capable of inverting sucrose rapidly, but are adapted to growth and development in sucrose solutions of the highest purity. A microbiological investigation of the potentialities of these species becomes one of the pressing requirements of modern sugar production and sugar refining, for it is more than likely that many of the species present, which are now capable of causing losses in sugars during manufacture, have acquired these properties by virtue of their long-continued exposure to the composition of the products in which they develop in sugar factories and sugar refineries. Recently, Owen and Bienvenu (14) isolated a species of bacteria and also one of mold fungi which not only grew readily a t thermophilic levels of temperature, but were also capable of producing rapid destruction of sugars in a medium consisting of raw sugar sirup of around 65 O Brix concentration. Until recently, controlled laboratory investigations of the ability of species of thermophilic bacteria occurring in sugar products to induce sucrose inversion or destruction within thermophilic ranges of temperature have not yielded consistent results. One of the greatest factors contributing to the failure t o induce development of microorganisms a t high temperatures in sugar liquors has been the conditions under which these tests have been made. A very essential point has been often disregardednamely, the catalytic action of colloidal material in the product tested. The function of finely divided suspended matter, especially activated carbon, Filter-Cel, bagacillo, or bone char, as an accelerant of the growth and activity of microorganisms has long been known. As early as 1913 Sohngen (16) observed that mch biocolloids as clay, filter paper, char, and earth very markedly accelerate alcoholic fermentation. Some years later, Abderhalden and Fodor (1) investigated the accelerative action of small quantities of bone char upon the rate of alcoholic fermentation of sugars by yeast. Ivekovic (9) conducted an extensive investigation of the effect of boneblack upon alcoholic fermentation and attributed its accelerative effect t o a.number of causes, among which is the absorption of toxic substances by the char, which minimizes the retarding action of the toxins upon the development of yeast. Owen and Denson (16),in 1929, found that small particles of finely divided bagasse, as well as activated carbon, not only accelerated the rate of fermentation
601
of sugars by yeast, but also enabled these microorganisms to withstand high temperatures and high codcentrations of alcohol. No. of Cells Surrounding Carbon Particles per
Sample Vegetable carbon Distillery oarbon
M1. of Sol 28 000 000
16:OOO:OOO
Av. No. of Cells in
Clear Space Per ml.
0.88 0.40
3,520,000
1,600,000
A review of the literature fully confirms the theory that microorganisms are able to induce fermentation of sugar solutions a t densities and a t temperature ranges far beyond the limits a t which they are ordinarily active, if sufficient inert colloidal material is present. These facts render suspect, therefore, bone char or vegetable carbon filters or the bagacillo in unclarified juice, before or during clarification. Prior to clarification cane juices contain a large amount of finely divided bagasse or bagacillo, which not only constitutes an ideal material for the protection of the organisms against high temperatures, but also accelerates growth of thermophilic organisms that are invariably present. During filtration over bone char or in vegetable carbon or in juices in clarifiers, the conditions are ideal for the development of microorganisms. Where losses occur in bone char filters, the explanation usually offered is that the chars are not completely burned during revivification, but present knowledge leads to the belief that in many such cases the sucrose losses are due to an accumulation of sufficient viable cells to induce rapid sucrose destruction. Bacteriological analyses of spent char from filters or activated carbon after the exhaustion of its adsorptive powers reveal the presence of a large number of thermophilic organisms. The number of thermophilic bacteria in some of these samples indicates that growth has undoubtedly taken place and there is a sufficient concentration to induce rapid sucrose destruction in the liquors passed over or through them. The presence of these adsorptive clarification agents is ideal for both the protection of the organisms against high temperatures and the acceleration of their development. Continuous passages of sugar liquor over or through these materials tend to concentrate both the organisms and the nutrient materials on the surfaces of the adsorbent and hence the microorganisms find ample nutrient material for their development. The detrimental effect of microbial growth upon the efficiency of sugar manufacturing or refining operations need not be entirely restricted to sucrose or loss in total.sugars. In many cases fermentative changes induced by microorganisms may be costly, independent of any sucrose losses. There are many instances in which refined sugar may have very slight, but objectionable, odors or tastes, as a result of incipient fermentation in the dilute liquors. In some cases there may be a very slight butyric odor as a result of butyric fermentation in the lighter liquors. Another condition which is assuming more and more prominence, especially in warm climates, is the presence of algae in water. supplies in the factory or refinery. Many of the algae form very undesirable odors in water supplies and some of the more common form fishy or sometimes aromatic odors. These odors are derived from minute quantities of oil contained in these plants and some of the odors are detectable a t dilutions as high as 1 ta 1,000,000. The writer has often found the water supplies used by the manufacturers of white sugar to be highly contaminated with algae. Where the water supply is kept in closed tanks protected from the sunlight, algae rarely occur, but it is often necessary to filter municipal water used for washing and boiling sugars through decolorizing carbons. Among the most prominent odor-producing algae is the “synura,” which is most prominent in waters in spring and autumn and has often caused bad odors in municipal water supplies. Whipple (18) found that as few as five to ten colonies per milliliter of water are sufficient to cause very perceptible and undesirable odors. In many cases the flow of water through pipes in distribution systems is sufficient to produce the
I N Q U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
608 Table I .
Sucrose Losses over Vegetable Carbon at 132' F.
for Various Intervals
(Sucrose solution over carbon for 5 days, then fresh solution introduced) Period of Incubation, Hours Brix Sucrose Purity 10' Brix Solution Control
18 24 48
10.00 9.60 10.10 9.50
8.40 8.00 8.40 7.00
84.00 84.21 83 17 73,68
60° Brix Solution
71 57 68.60 54.83 36.95 52.50 These analyses were made on successive portions of sugar solutions o r eirups, exposed for varying periods of time t o contaminated carbon, rather than the same solution, exposed for varying tlme intervals. Control
60.50
24 48 5 days
60,50
60,60
43.30 41.50 32,QO 19.40
disintegration of many forms, and the odor of a water a t the service taps is more pronounced than a t the reservoir. The odor is often intensified by the addition of salt or sugar solution; hence an originally imperceptible odor may become pronounced in the sirup or sugar on nhich it is used. Whipple has claimed that a water containing 100 colonies of synura per milliliter might produce an odor after a dilution of 1 to 25,000,000.
DETERIORATION OF SIRUPS AND MOLASSES AS A RESULT OF MICROORGANISMS The loss of sucrose in manufacturing, as a result of the activities of microorganisms, is not entirely confined to juices or sugar liquors of low density, for in many cases considerable losses occur in sirups. Cane sirups are very subject to fermentation by yeast, especially by Torulae, which as a rule do not invert sucrose, but there are occasions when this fermentation results in a very high and very extensive loss of sucrose. De Whalley and Scarr (6) reported an incident where raw refinery sirup, which was stored while hot, contained a very high infection of Torulae and 11% of alcohol was produced as a result. .4s a rule, the fungi are the more consistent and prevalent microbial agencies responsible for sucrose losses in either sirups or molasses, and the fact that raw sugars with high moisture rontent, which have a "factor of safety" of over 0.33, decrease rapidly in polarization s h o w that the mold fungi can exercise inverting action a t high ranges of density. Although blackstrap molasses is very prone to undergo changes which in every respect resemble true fermentation, this is an autogenous change, due to spontaneous decomposition. This subject has been extensively investigated by Zerban (19), Hucker and Pederson ( 8 ) ,and more recently by Owen ( I S ) , who found that whereas the total sugar losses were high in almost every case, this loss was c o n k e d to the reducing sugars already present. I n order to determine the rate of sucrose destruction resulting from the activities of thermophilic microorganisms, the following experiments were carried out: Twenty grams of a finely divided, activated carbon and one of a coarser mesh, such as is used in water purification, were introduced into 200-ml. bottles, and approximately 50 ml. of a sugar solution, which had been heavily inoculated with thermophilic bacteria obtained from sugars, were introduced over the carbon. The amount of liquid added was just sufficient to maintain a level of approximately 1 inch above the surface of the carbon particles. These bottles were then placed in a thermophilic incubator and incubated at a temperature of 132" F. for 4 to 5 days. The length of this initial exposure was largely determined by the concentration of bacteria in the liquid, as shown by microscopical examination of the solutions. When this concentration reached its desired level, which never required over 5 days, 100 ml. of freshly sterilized sucrose solutions were added to each bottle. The samples pipre placed in the thermophilic incubator and the liquid then was withdrawn from the container for an analysis, by means of a sterile pipet. B s soon as all the liquid had been withdraan, a fresh portion of the sterile substrate was introduced and the bottles and contents were incubated for successive periods.
Vol. 43, No. 3
Table I shows that the loss in purity of a 10 O Brix solution was progressive throughout the entire period, 48 hours, and the purity had dropped from 84 to 73.68 in that time. Control samples of the inoculated sirups or solutions were exposed in the incubator for the same length of time, in order to determine the effect of microbial action in the absence of carbon. A similar experiment using a sucrose solution of approximately 60' Brix was carried out, with the carbon inoculated with thermophilic bacteria from the previously exposed samples. The deterioration of the solution in contact with carbon amounted to an approximate drop of 35" in purity during a $day incubation period. Microscopical examinations of these exposed solutions, which had undergone deterioration during storage at thermophilic levels of temperature, revealed that the concentration of bacteria was very high, but all efforts to isolate them under aerobic conditions proved futile, as few or no colonies developed on the substrate after an incubation period of from 4 to 5 days. Under anaerobic conditions, where the media were placed in deep levels over the carbon, the development of microorganisms was noted by a vigorous evolution of gas, which was sufficient t o cause gas pockets throughout the entire medium. It is evident that the bacteria growing under these conditions and responsible for the loss of sucrose are anaerobic thermophilic bacteria, but methods for their enumeration have still to be elaborated. Similar experiments were made with boneblack instead of activated carbon, using 20 grams of the char per 100 ml. of the sucrose solution to be tested. These experiments were carried out on both 10" and 60" Brix sucrose solutions (Table 11).
Table 11.
Experiments w i t h Boneblack Control Carbon Sucrose Solution
Brix Suorose Purity
10.00 8.40 84.00
Brix Sucrose Purity
45.30 75.00
60,46
18 Hours 9.60 8.00
24 Hours 10.10
84.21
8.40 83.17
63.90
61.50
47.70 74.65
42,60 69.27
48 Hours 9.50 7.00 73.68 61.40 39.91 65.00
The drop in purity of both the 10" Brix and 60" Brix solutions during an exposure of 48 hours to a contaminated char amounted to approximately 10.5'. I n order to determine the effect of the addition of a bacteriostatic principle to carbons, as a means of preventing microbial growth and the sucrose destruction incident thereto, the same carbon which was used in the previous experiments was treated in this manner. A dilute solution of the bacteriostatic principle was percolated over the carbons, which were dried in an oven a t 130" F. The dried carbons were then impregnated with the thermophilic bacteria obtained from previously deteriorated sucrose solutions used in earlier tests. The control and the treated carbons received identical inoculations with this material (Table 111). While the contact of the solution with both the control and the treated carbons (Table 111, A ) induced sucrose losses, the purity of the solution exposed to the treated carbon was from 3 to 4 degrees higher in every case than the solution exposed to the untreated material. The values for purities in the samples after different periods of incubation do not decrease in a synchronous order-in some cases the samples after longer exposures show less drop in purity than those incubated for shorter periods of time. This apparent inconsistency may be explained by the fact that the analyses over the successive periods of incubation were made upon replicates of the original solution and not upon the same sample at successive intervals of time. The purity of the sucrose solution in contact with the treated carbon (Table 111, B) remained practically consistent a t 81.63 to 81.73 during the 48-hour incubation period, which represented
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Table 111.
.
Treated Carbons Impregnated with Bacteriostatic Material Treated Con24 Control Hours trol 9 . 9 0 10.20 11.20 7.40 8.78 7.90 74.75 77.45 77.68
Treated Treated 48 Con72 Hours trol Hours 10.40 ... ... 8.50 ... . .. 81.73 .
A.
Brix Sucrose Purity
Sucrose Solution 10.40 8.80 84.62
B.
Brix Sucrose Purity
10.40 8.80 84.62
9 . 8 0 11.20 10.40 9.90 7 . 4 0 8.00 8.78 8.50 74.75 81.63 77.68 81.73
C.
Brix Sucrose Purity
10.07 9.70 96.32
13.04 14.25 11.15 11.90 10.40 10.56 10.20 12.60 9 . 5 0 10.20 9 . 5 0 10.00 78.22 88.42 85.20 91.89 91.35 94.70
. . . . .. .. ..
... .. .. . ... ,
a loss of purity, as compared with the control, of approximately 3”. In contrast to this loss was the drop of IO” in 24 hours, in purity of the solution in contact with the control carbon. The maximum deterioration of the sucrose solution which was in contact with the control carbon (Table 111, C ) was approximately 18”’while the maximum loss in sucrose for the treated carbon was only approximately 8 ” . In both instances the greatest deterioration occurred in the samples that had been exposed only 24 hours. These results can hardly be regarded as entirely parallel to conditions obtaining in sugar manufacture, for only in the rarest instances would sugar liquors be exposed for so long a time to temperatures as low as 132” F. Nor would there be many instances in which sirups or sugar liquors, containing any of the colloidal decolorizing materials, would be held over for any length of time within the range of activities of thermophilic bacteria; but there is a real possibility of this condition’s obtaining in the distribution lines of factories or refineries, a t times some modified deteriorative action of this kind may occur during standard operations of char or activated carbon filters. It is the writer’s opinion that many cases of undetermined losses in sucrose may prove to be due to the action of thermophilic bacteria under conditions somewhat similar to the ones maintained in these laboratory tests. Certainly, where these losses are unusually high, the results herein reported should a t least tend to render suspect the known potentialities of thermophilic bacteria to induce the destruction of sucrose. From these results obtained from the preliminary stages of what is planned as a full and complete investigation, the author feels warranted in drawing the following conclusions. CONCLUSIONS
The preliminary survey of the potentialities of thermophilic microorganisms to induce rapid destruction of sucrose in solutions of the densities comparable to those of cane juice and first
609
liquors in refineries, indicates that to this action may be attributed many instances of undetermined sucrose losses in sugar refining. The accelerative action of both bone char and activated carbon, as shown in these preliminary results, is indicative of the role that contaminated filtering material progably plays in contributing to the undetermined losses in sugar refining. The proved ability of thermophilic anaerobes to develop in sucrose liquors within the temperature ranges to which these liquors are not infrequently exposed in practical operations may explain the sporadic occurrence of large numbers of these microorganisms in sugars manufactured for the canning trade. This investigation is being continued under conditions more closely approximating those of plant operations, and in the hope of more completely exploring the full extent of this potential source of sucrose losses in refining operations. LITERATURE CITED Abderhalden, E., Fermentforschung, 5, 90-110 (1921). Cameron, E. J., and Bigelow, W. D., IND.ENQ.CHEM.,23, 1330 (1931).
Cameron, E. J., and Williams, C. C., Centr. Balct. Parasitenk., 11, 76, 28-36 (1928).
Carpenter, C. W., and Bomoqti, H. F., Hawaiian Planters Record, 24, No. 5, 198-203 (1920). Cienkowski, L., “Die Gallertbildungen des Zuckerrubensaftes,” Charkow, 1898. De Whalley, H. C. S., and Scarr, M. P., Chemistry & Industry, 1947, 531-6.
Diago, R. E., and Guerrero, F., Sugar, 42, No. 11, 30-2 (1947). Hucker, CI. d., and Pederson, C. S., Food Research, 7, No. 6, 459-80 (1942).
Ivekovic, H., Biochem. Z.,183,451-60 (1927). McAllep. W. R.. and Walker. H. S..Report of Committee on Juice- Deterioration, 18th Annual Meeting of Hawaiian Chemists’ Association, with Hawaiian Engineering Association, November 1920. McCleery, W. L., “Deterioration of Cane Mill Juices from the Aspect of Acidity Increase,” Third Annual Meeting of Association of Hawaiian Sugar Technologists, Honolulu, Oct. 27. 1924.
Owen, W. L., Facts About Sugar, 16, 519, 521 (1923); La. E z p t . Sta. Bull. 11, 162.
Owen, W. L., Sugar, 38, No. 12, 22-4 (1943); 39, No. 1, 22-4, NO.2, 24-7, NO. 3, 26-9, NO. 4, 26-9 (1944). Owen, W. L., and Bienvenue. R. J., Intern. Sugar J.. 51. 22-4 (1949).
Owen, W. L., and Denson, W. P., Centr. Bakt. Parasitenk., 11, 77, 481-523 (1929).
Sohngen, N. L., Centr. Bakt. Parasitenk., 11, 38, 621 (1913). Van Tiegham, P. E., Ann. sei. nat. Botan., 6 , 180-202 (1878). Whipple, G. C., “Microscopy of Drinking Water,” 2nd ed., New York, John Wiley & Sons, 1905. Zerban, F. W., “Color Problem in Sucrose Manufacture,” New York Sugar Research Foundation, Technol. Rept. Series, No. 2 (August 1947). RECEIVED April G , 1950.
Samples of Sugar Juice Are Taken from Vacuum Pan to Determine When Crystals Have Reached Their Proper Size
