Semi-Plant Scale Production of Gluconic Acid by Mold Fermentation

Semi-Plant Scale Production of Gluconic Acid by Mold Fermentation. O. E. May, H. T. Herrick, A. J. Moyer, and R. Hellbach. Ind. Eng. Chem. , 1929, 21 ...
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INDUSTRIAL AND ENGINEERING CHEMISTRY

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consistency exists for dispersions of the zinc-oxide and linseedoil vehicles used in these experiments. Explanation of Results The oil-absorption values of the pigment for the different oils and the consistencies of the spatula-mixed pastes are dependent upon two opposing factors-degree of wetting and soap content. An increase in the degree of wetting reduces proportionally the oil-absorption values of the pigment for the different oils and the consistencies of the spatulamixed pastes. An increase in the amount of soap formed causes an increase in these properties. A neutral or very low acid oil does not readily wet the pigment because of a film of adsorbed moisture. A small increase in acid number of the oil causes a marked increase in the degree of wetting with the formation of only a small amount of soap. The result of these opposing factors for a slightly acid oil is a decrease in the oil absorption of the pigment and in the consistency of the spatula-mixed pastes. The increase in degree of wetting with increase in acid number approaches a maximum a t an acid number of 5 or 6. Further increase in acid number does not cause much increase in degree of wetting, but the amount of soap formed increases continuously as the acid number is increased. Therefore, the oil absorptions and consistencies of the spatula-mixed pastes which result from the use of linseed oil with an acid number slightly above 5i. e., from 5 to 15-are almost constant, because the two opposing factors of wetting and soap formation, for oils of these acid numbers, approximately balance each other. As the acid number is increased still further, the effect of soap formation relative to degree of wetting becomes increasingly greater. The oil-absorption values of the pigment and the consistencies of the spatula-mixed pastes increase, therefore, more and more rapidly as the acid number of the oil increases.

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The consistencies of the ground pastes, on the other hand, are not appreciably affected by the degree of wetting of the pigment by the oils, because the energy supplied in the grinding process is sufficient to cause the oil to displace the film of adsorbed moisture and wet the pigment. The primary factor which influences the consistencies of the ground pastes is the amount of soap formed in the paste. Since an increase in acid number causes increase in soap concentration, and an increase in soap concentration causes an increase in consistency, the consistencies of the ground pastes must increase continuously as the acid number of the oil increases. Almost any relationship between oil absorption and consistency can be shown to exist by an inspection of Plate 111, if the conditions for obtaining the experimental data are properly chosen. For example, if one oil with an acid number of 5 and another with an acid number of 20 were chosen as the vehiclcs to be used with the zinc oxide employed in these experiments, an oil-absorption value of approximately 12 would be obtained in each case. The apparent viscosities of the ground pastes, however, would be, respectively, about 120 and 220. The difference in apparent viscosities is probably due to the unequal concentration of soap in the two pastes. Since different amounts of soap-forming constituents are known to exist in different types of lithopone, it is possible that the results obtained with the lithopone pigments are due to differences in the amounts of soapforming materials present in the samples. Lack of information a t present in regard to the chemical composition of these pigments prevents testing this hypothesis on these samples. The study will be continued on specially prepared lithopones. Literature Cited (1) Baldwin. IND. ENG.CHEM, 21, 1121 (1929).

Semi-Plant Scale Production of Gluconic Acid by Mold Fermentation' 0.E. May, H. T. Herrick, A. J. Moyer, and R. Hellbach COLORA N D FARM WASTEDIVISION, BUREAU OF CHEMISTRY A N D SOILS, WASHINGTON, D. C.

HE formation of glu-

Gluconic acid has been produced on a semi-plant quantities of citric and oxalic conic acid as a result acids were also formed. Bernscale by the action of Penicillium luteum purpuroof mold metabolism gefwm on solutions of commercial dextrose. Studies h a u e r , however, has develwas first reported by Molliard have been made of the effect of surface-volume ratio, oped a strain of Aspergillus in 1922 ( I O ) . In the course subsurface agitation, and PH. A high grade of aluminiger which, in the presence of an investigation on the pron u m has been established as a suitable material for ferof calcium carbonate and unmentation Pans. Using Seven aluminum Pans, 43 by 43 duction of citric and oxalic acder definite conditions of culids by a culture of Aspergilby 2 inches (108 by 108 by 5 cm.), about 36 kg. of gluconic ture, yields gluconic acid alZus niger on 5 per cent sucrose acid have been obtained on a n Il-day cycle, a yield cormost exclusively (3). solutions Molliard noted that I n the course of a survey of responding to slightly more t h a n 57 per cent of theory. the total acids recovered as the action of a large number of insoluble calcium salts from the fermented solutions did fungi on solutions of commercial dextrose it was found that an not account for the total titratable acidity. A third acid organism of the Penicillium luteum purpurogenum group was which he then isolated from the mother liquors was capable of oxidizing this sugar to gluconic acid in promising identified as d-gluconic acid from the properties of t h e yields without the formation of other acids (9). Further calcium and cinchonine salts, and the phenyl hydrazide, and study of some of the variables affecting the fermentation, by elementary analysis. Within the last five years Butke- such as temperature, concentration of dextrose, and composiwitsch ( d ) , Falck and Kapur ( 5 ) ,Bernhauer (Z), Wehmer ( I d ) , tion of the nutrient salts, resulted in increasing the yield to Takahashi and Asai (IS),and Amelung ( I ) have reported around 60 per cent of the theoretical and demonstrated that finding this acid among the products resulting from the mold even under widely varying conditions of culture no apprecifermentation of solutions of dextrose, sucrose, and maltose. able quantities of other acids were formed (6). In most cases the yields were comparatively low and varying I n 1919 Herzfeld and Lenart suggested that gluconic acid * Received October 25. 1929. might prove a valuable addition to the group of aliphatic

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a d s na:d in industry and outlined a process for its cominc~rcia1 production hasod on the oxidation of dext.rose i&li bromine ( 7 ) . The process was unsatisfnctory and was moilified lip Ling and Nanji so that the qnantity of bromine rcqriired was considerably redued (8). The modified procediire was still somewhat objectionable from tlic industrial staridpoint, as a large proportion of tlic bromine which was still nceded could not be recovered for further use. Rcoently p t ents h n r c been granted co for the nianufactiire of t,ha neid from dextrose based i i p n the use of 1iypiichloriti:s with small quantities of nlkali iodides or bromkles as oxidizing agents (11, 12). The acid is now being manufactnrrd nmlrr these patents, nltliougli no production data are available.

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diffusion and tilt: products of oaidatioii be distribnted thronghoiit the solution in tlie same vay. It v a s tiioiiglit, tliiwfore, that agit,atioii of the substraturn might d rate of reaction nnd licnre promote 11 in a given t.ime by priwntin!: tlie maxiininn conr:emtrntioii of sugar to the r i i j linsii at, i ~ l times l and also by prevent,ing mi accumulation of acid iiircntly beneath it. Sucli agitation niiglit also permit, the economical use of larger vt~liisnes of solntion or liigiicr coiicentrations of dextrose. Since a quiescent inyceliuni is absolut,clyessential for a normal wncntiition, as sul~mergenceof evcn a portion soon C R U S ~ S k e wliole mat to sink with complete cessation of oxidation, an apparatus which would agitate the substratnm thoroughly without disturbing the surface growth was devised. The details of r:onstruct,ion are given in Figure l . A series of ex.. perimciits was carried oiit with solutions containing 100,150, 200, and 250 parts of commercial dextrose per liter of solution. TIic surface area-volume ratio was 0.28. To maintain nniforin conditions a flask containing a stationary agitator IVW used as a control in each exmriment. The averaee vields of two series of runs for each concentration are given in Table I. ~ : d i i l l i ib y

~II

-

Table I-Effect of A*IrarIon

I.

FiCvre 1 -ABifacion Device

l l i e lrigli .yields of acid given by I'enicilliuwL luteum purpuroqcnunz seemed to justify an investigation of its industrial prodnction by fermentation, in view of the relatively nnsatisfactory state of tlie strictly chemical processes. The biochemical process is fairly easily controlled, yields no troublesome by-products, and permits a rcady recovery of the pure acid as the calcium salt, or as a crnde acid containing residual dextrose, by evnporation and char treatment of thc fermented liquors. As tlrerc is nothing i n the literature concerning the largo-scale! application of a proccss involving the utilization of a mold mycelium resting on the siirface of a liquid substratum. it was felt that any information obtained in this investigatioii would be of value in other processes of a similar nature.

CVMVLBRCIAL DBXTROSE

Pes 1000 cc. SOLN.

G*omr 100 150 200 250

-----YIELOI-----

contio1 Per rcnl 27.8 49.2 55.0 44.7

Agilricd SUI". Per cent 40.5 66.8 80.0

44.7

The resuits in Table I ilidieate that the effect of agitatiori on acid prodnetion decreased as the concentration of sugar was increased. With the lower concentrations stirring has a

Surface-Volume Ratio

The ratio of the surface area of the inycelium t o tlie volumi, of solution being fermented is an import,ant factor in any ferinentation of the type under discussion. The yields of gluconic acid obtained have varied from 82 pcr cent of theory (almost the maxininin yield possible), when tliis ratio, EXpressed as sq. cm. per cc., a.pproached uiiity: to 30 per ccnL of throry, when its value was 0.16. The percentage yield is roughly proportional to the ratio, but, of coi~rsc,from a certain point the actual might of acid for a given area of rnycrlium is much l o ~ w with r sniallm volnmes. For practical purposes it was found best to rmploy such volumes of solutioii that the ratio lay between 0.25 and 0.30. In that range arid with concentrations of commercial dextrose of from 20 to 25 pcr cent, yields of from 55 to 65 per cent of thc tireoretical liave consistently resulted and t,he maximum actual weight of tlie acid has been obtained. From the results of large numbers of experiments in which sucli values for surface area-volume ratios and sngar concentrations were nsed it. appears that each square meter of mycelium is capable of yielding from 4 to 4.5 kg. of gluconic acid in 14 days from the time of inoculation from spores. Subsurface Agitation

The forrnatioii of the acid takes place entirely within the cells of the mycelium. It is obvious that under ordinary circumstances the dextrose in the solution must reach the my-

Fitkure 2-Sfoik

Oulrures and Laboratory Fcrmenfafions

iiiarkcii effi!i!t hit,, apimently, tlie inasirnmri quantity of dextrose wliich the organism has the ability to oxidize reaches the mycelium by diffnsion in the 20 and 25 per cent solutions. However, tlie quantity of acid proiiuced in the liiglier concentrations is sufficient to offset any advant,age gained by stirring

in the lower concentrations. These experiments eonfirmed the opinion that a shallow-pan set-up was the only feasible nianner in wliicli this fermentation could be carried out. Hydrogen-Ion Concentration

Some experiinents on the effect of hydrogen-ion concentration, in wliicli the initial pH ranged from 6.4 to 3.0, have been condncted. In tlie first series the pII of the usual 20 per cent commercial dextrose solutions containing the necessary nutrient salts was measured after sterilization and cooling. It was found t o be 4.8. I n the second series 10 grams of sterile

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calcium carbonate wereadded to tlie solution after sterilization and cooling. AFh?r t,lie flask was shaken tlioroughly and allowed to stand for a short time the pH was 6.4. In the third series a small quantity of gluconic acid was added to give an actual pH of 3.0. TIie solutions were inoculated a i d the fermentation was allowed to proceed for 14 days. It, was necessary to shake the flasks containing the calcium carbonate gently tvice daily until the carbonate disappeared, to prevent tlie developmrnt of aii acid solution directly beneath the mycclium. The results of these experiments are suriimarized in Table 11.

.

Tahle II-~~~EKect Of Hydroflen-Ion Concentration INlrilL SOLUTION

Z'IZ

FINAL

PIr

ACID POPIMIID

C"""," -.I.."

DPxtroie S d U t i O " ~ 4.8 2 08 Dextrose sOIU,ion~ C U I C i l i l l l carhonrte 8.4 3.0 Dcxirore rolufion" gluconic acid 3.0 2.06 Ordinslry 20 per cent carnrnercirl dextrose SDLU~IO~.

++

Figure 3-Enamel

117.2

125.0 120~7

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Table Ill-Fermentation COMMSRCldl. DBXTXOSB

GslProurio

KZ. 8.0 8.0 8.0

In Enamel Pan PlmR D B I I P \ O S R RHMAENACID SORXUD 1x0 U X P B I I B N T ~ D YIXl.0 KI. % oflheory 4.8 1.5 64.0 t.7 1.7 58.7 4.9 1.4 6.1.2

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r, sevcral objections to the use of such pans on an indiistrial scale. They are heavy and unwirldy and also expensivc. If the enaniel is chipped or cracked Lhrough careless handling the pails become useless and have practically no scrap value. Attention was next turned to the 11se of pyroxylin lacquers sprayed on sheet-iron pans. Experiments with small pans made up in this manner sliowcd that. a normal fermontat,ion c d d be carried out in them. Wheii large pans isere used some t,rouble was experienced with blist,ering of the coating in tlie course of steam sterilization. This, however, was overcome to a large extent tliroiigh proper preliminary treatment of tlic inetal and use of the riglit type of undcrcoat,cr. Several fermentations were carried out in large pans of this typc with satisfac,tory yields of acid, and tho problein of pan matcrial was considered solved. However, it was ohscrved, after a number of runs, that the lacquer began to elmirge in color from white to a rather deep brown, accompanied by a decided drop in acid production. Continued use of these pans finally resulted in yields of acid in the neighborhood of 30 per cent of the tlieoretical. That the organism was heiiig affected was indicated by ita appearance. Instead of a normal, smooth, faintly yellow, and durable mycelium, a roughen~d,grayish, and brittle mat was the rule, while controls run in flasks at t,he same time showed normal development. This pointed to the formation of some toxic decomposition product of the lacquer in the course of st,erilization and fermentation. On this accouiit the use of the cheap and conveoicnt spray lacquer for a coating material for the pans liad to bi: abandoned.

and AIuminum Pans

BY no attempt was made to prevent changes in p1.I during the fermentation, the results are somewhat inconclusive. I n all cases, however, there was no marked difference in the rate of growth or in the appearance of the mycelia, and the final qiiantity of acid formed indicates that hydrogen-ion concentrat.ion over the range investigated bas no marked effect on t.he oxidation.

Choice of Pan Materials

With t.hese factors established the successful transfer of this fermentation from a laboratory to a semi-plant scale depended chiefly on finding a suitable material for construction of the pans. The chief requirements t,o he fnlfillcd were resistance to corrosion by tlic acid, non-toxicity t,othe organism, low cost, and durability. Preliminary experirnenis indicated that nickel, lead, copper, and monel metal, while acid resistant, were markedly toxic to the organism. Zinc, iron, and, to some extent, the ordinary grades of aluminum were dissolved by the acid. Block tin and bakelite were also tried and proved fairly satisfactory, but were eliminated from consideration on the ground of cost. The introduction of nentralizing agents such as calcium carbonatc into the solution would have necessitated agitation of some sort, which n-ould liavc unduly complicated a shallow-pan process. Ordinary cheap enaniels were completely dissolvcd by the acid. A small iron pan coated with high-grade acid-resistant enamel in which fievwal runs could be carried out with entire sueccss was finally obtained. Many fermentations of 40 liters of solution were then conducted in a larger pan (40 by 40 by 2.5 inches) of the same material, with yields equal to tiiose obtained in glass. The results of some representative runs are given in Table Ill.

Figure 4-Typical

Pan Fermentation

While ordinary commercial gradcs of aluminum were not satisfactory for pans, further experimentation with material conhining a t least 99.45 per cent of ahiminum and less than 0.1 per cent of copper and manganese gave promising results. Losses by corrosion on tcst samples could not he deteetcd by ordinary met,liods of weighing. Small pans of such material permitted a normal development of tlie orgaiiisin and, while Riving only 40 per cent yields in the first two runs made in them, ahowed marked improvement as soon as a doll coat.ing had formed on the int.erior surfaces. Pans 43 by 43 by 2 inches (108 by 108 by 5 em.) made of this material were accordingly bought, and, aft.er a preliminary treatment nrith hot dilute gluconic acid, two runs were macle. The yield

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was a.round 45 pix cent. of theory. Following this, four successive fermentations gave yields from 52 to 61 per cent of theory. I t is felt that such pans are suitable for fermentation, being light, durable, and fairly cheap, especially when ordered in large lots. Moreover, they have a scrap value which represents a large proportion of the origiiial cost.

has been checked. A good colony on peptone-dextrose a t the age of 10 days is usually 5 to 6 em. in diameter and has a light blue-gray color, except on the outer edge, wliich is white wit11 a trace of yellow. Such colonies have a more luxuriant vegetative development and less sporulation than strains of penicillia commonly found on decaying frnit.

Fieure 5---Seven-Pan Instnilation in Operation

Figure 6-Pronf of A p a ~ ~ f uShowing e, Peep Holes s n d Inoculatfon and Filling Tubes

Maintenance of Organism

The selection and careful mainteiiance of a pure and productive strain of organisin are essential features of this work. During the last two years cultural descendants of the original fungus have shorvn considerable variability. Strains which differed in colony coloration, vegetative vigor, degree of sporulation, and acid-producing power have appeared. Such a situation has made it neeessary to inake careful cultural tests of this organism. By using small flask cultures, it has been possible to test several apparent mutations and select a high acid-producing strain. All the condit,ions that cause or prevent tlie degenerat.ionof stock cultures are not clearly understood. However, a procediire which seeins satisfactory in maintaining tlie desired biochemical and vegetat.ive characteristics of the organism hasbeeii adopted. Wort-agar slanted in test tuhcs supports a vigorous growth of the fungus and does not react unfavorably OD its acid-produring capacity. When preparing agar cultures lor imtnediate use, t.ransfers are niade from a tested stock culture on wort-agar to another solid medium of tlie following composition: 13iacto-Iieptone "Difco," 15.0 grams; commercial dextrose, 30.0 grains; agar-agar, 30.0 grams; distilled water to make a total volume of one libcr. Such a mediuni favors a good vegetative growth and a uniform and heavy sporulation over the entire slant in 8 to 10 days, when incubated at 25' to 30" C . The ratlier high agar colicentration facilit,atestlie reniural of spores and mycelium during the inoculation proeediirr. All t.hc stock cnltures are stored at 4' C. a.nd have reninincd in a desirable condition much longer tlian others kept a t room temporature and room humidity. To detcrniirie the possible prcseuce of mutations and eontaminations, spore dilutions have becn plated as a matter of routine and the resulting colonies observed for variations. Also fragments of mycelium from stock cultures have been transferred to agar plates and the regularity of tlie colonies

When tile procedure outlined is followed little trouble, if any, need be anticipated in maintaining a pure and vigorous acid-producing strain of the fungus. Apparatus Set-Up

A rack liolding seven of the large aluininunr pans, one above the other, with a 4.5-inch clearance between them, was constructed from angle iron. The framework was coated with a high-grade varnish to prevent rusting. The rack was installed at one end of a small constant-temperature room so that the walls protected it on three sides. A steam line was led into the room and terminated under the center of the bottom pan. The front of tlie apparatus was closed in by a removable framework of wood, over which four tliicknesses of clieeseclotli w'ere stretched to permit diffusion of air and to prevent eontainination by foreign fungal spores. 13acteria and yeasts do not thrive under the conditions of this fermentation and thus far have caused no trouble. The front was fitted with peepholes to permit observat.ionof tlie progress of the fermentation and also wit11tubes leading to each pan for inoculating and filling. A fan blower supplkd sterile air to the pans through a conduit leading to two vertical perforated pipes at tlie back of the pans. Thermometers, which could be read tlirougb the pceplioles, were suspended horizontally above each pan. Above the level of the top pan was installed a reservoir from which tlie sugar solution was ineasured into each pan through a wrought-iron pipe connected to the filling tubes by rubber tubing. Fermentation Procedure

Three days before tlie start of a run the inoculation flasks were made up. Twenty-one flasks, each containing 400 cc. of solution of the same composition as that used in the fermentation, were sterilized for 15 minutes at 15 pounds steam pressure. These flasks were inoculated from agar-peptone-

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dextrose slants of pure cultures of a tested strain of the or- and allowed to crystallize. When cold it was centrifuged, ganism, a t least 10 days old, one tube being used per flask. washed with a small quantity of ice water, and dried a t 80" C. On the third day after this inoculation, the sugar solution to The filtrate was returned to the neutralized fermented liquors be used for the fermentation was made up. For each run of for further concentration. The calcium gluconate recovered seven pans the following quantities of material were required: at this point, if the crystallizations are properly carried out, commercial dextrose (approximately 91.5 per cent of dex- is satisfactory for general use. When used for pharmaceutical trose), 63 kg. (139 pounds); sodium nitrate, 0.315 kg. (0.7 purposes a further crystallization may be advisable. Glupound) ; MgS04.7H20,0.079 kg. (0.17 pound) ; potassium chlo- conic acid was obtained by adding the calculated quantity of ride, 0.016 kg. (0.035 pound); phosphoric acid (100 per cent), dilute sulfuric acid to a hot 15 to 20 per cent solution of cal0.009 kg. (0.0198 pound); water to make 315 liters (83.2 gal- cium gluconate. After the calcium sulfate was filtered off lons). the acid solution was concentrated. The liquor may be deThis solution was sterilized by heating in a steam-jacketed ashed with barium chloride a t this point, if the presence of autoclave to 100" C. The autoclave was connected to the calcium chloride in the liquor is preferable to that of calcium reservoir by a wrought-iron pipe, and this line, the reservoir, sulfate. This concentration may best be carried out by and the line from the reservoir to the pans were sterilized by vacuum evaporation. The acid usually acquires a light yelpassing live steam through them for about 45 minutes. low color, which can be removed by a char treatment of the Simultaneously the steam line leading into the fermentation hot solution. It is not advisable to carry the concentration chamber was turned on and the pans and chamber were much beyond 80 per cent of gluconic acid by weight, as a steamed thoroughly for from 4 to 5 hours, after which they considerable quantity of lactone, which may crystallize sponwere cooled to 25" C., the optimum temperature for the fer- taneously on standing, is thus formed. A crude gluconic acid mentation. When the sugar solution had been cooled to suitable for many purposes may be prepared by concentration around 30" C. it was blown to the reservoir by sterile com- of the original fermentation liquor. I n this case the liquor pressed air. Three of the 3-day old cultures in the inoculating is given a char treatment a t the boil, filtered, and concentrated flasks were poured into each pan, and 45 liters of sugar solu- under a vacuum to a sirup containing approximately 30 per tion were added, thoroughly distributing the inoculum to each cent of gluconic acid and 12 per cent of dextrose. The data of a representative fermentation are summarized part of the container. I n 2 days a continuous film of mycelium which thickened rapidly was formed. On the fourth in Table IV. The values given are the averages of those obday a rise in temperature was observed, sometimes as much as tained in the last four runs with the 7-pan apparatus. 5" C. during the fourth and fifth days combined. Through Table IV-Normal 7-Pan Fermentation titration of samples of fermenting solutions it has been estabFERCOMSUGAR SUGAR WEIGHT UNOF lished that gluconic acid formation then begins a t a rapid MENTATION MERCIAL ACID FOR PERIOD DEXTROSE FORMEDACID FERMENTED MYCELIUMYIELD rate. On the fourth day the fan supplying air, cooled to 23" Days Kg. Kg. Kg. Kg. Kg. % of theory C., a t the rate of 3 cubic feet per minute, was started. This 11 63.0 36.2 33.1 16.8 1.25 67.4 volume of air was sufficient to furnish the necessary oxygen The fermentation cycle may be shortened several days by and also to hold the temperature above the pans to 25" to 28" C. At about the sixth day fine droplets of exudate ap- allowing the organism to develop in the inoculation flasks for peared over the entire surface of the mycelium, attaining con- a longer period or by emptying the pans earlier. The acidsiderable size (3 to 5 mm. in diameter) by the tenth day. production curve shown in Figure 1 in a previous paper (6) These droplets consisted chiefly of gluconic acid solution, no answers the second of these two possibilities, but it was traces of reducing sugars being present in them. The pans deemed advisable to investigate the first one further. Exwere emptied on the eleventh day. The mycelia were pressed periments in which pan inoculations were made from 4-, 5-, out and thoroughly extracted with hot water and the extract and 6-day old flask cultures, the time of pan fermentation bewas added to the fermented liquors. The volume of solution ing 10, 9, and 8 days, were conducted. The results are given was measured and analyses were made for acid and dextrose. in Table V. The mycelium was dried a t 60" C. for 3 days and weighed. Table V-Effect of T i m e Factor on Gluconic Acid Production These dried mats contained approximately 45 to 50 per cent FERCOMSUGAR S U G A R WEIGHT MENTATION MERCIAL ACID FOR UNOF of carbon and 2 per cent of nitrogen. The fermented solution PERIOD DEXTROSE FORMED ACID FERMENTED MYCELIUMYIELD was neutralized with either calcium carbonate or slaked lime, 0.98 39.1 22.5 25.0 8 63.0 24.5 calcium carbonate being preferable. If slaked lime is used 21.2 1.10 46.7 26.9 9 63.0 29.3 care must be exercised to avoid excessive decomposition of 18.1 1.20 53.5 30.8 33.6 10 63.0 the residual dextrose. The lime must be thoroughly slaked It is thus seen that any shortening of the fermentation cycle and added slowly, with effective stirring, to the cold liquors. It is advisable to keep the pH below 4.5 a t all times. The involves a loss of gluconic acid production but, of course, neutralization should be completed with calcium carbonate. permits a more frequent use of the fans. Costs for the production of gluconic acid and the gluconates The solution was then evaporated to s!ightly less than onehalf its original volume to give a 25 to 30 per cent solution of by this process depend largely on the volume of production. calcium gluconate. On cooling, the solution set to a Reliable figures cannot be determined from the operation of solid mass of mushy consistency, from which the calcium a unit of the size outlined in this paper, but some idea of their gluconate was separated by filtration or centrifuging. The magnitude may be obtained. A comparatively large initial filtrate, which contains practically all the residual dextrose, investment is necessary for the pans and installation. The coloring matter, and some calcium gluconate, may be given cost of raw material ranges from $4.00 to $5.00 per 100 pounds, a char treatment, and, after addition of nutrient salts and which, based on a conservative yield of 50 per cent, brings the making up to proper concentration of dextrose, returned to cost to $8.00 to $10.00 per 100 pounds of acid. However, the fermentation process, although it is advisable to keep the a t least one-half of the dextrose lost should be recovered by reworked sugar in a separate part of the process and to discard a secondary fermentation either to gluconic acid or to alcohol. the mother liquor occasionally, to avoid accumulation of im- The cost of nutrient salts is very small, not more than $0.05 purities, The crude calcium gluconate was dissolved in water per 100 pounds of acid. Plant operation costs would vary to give a 25 per cent solution, heated to boiling, filtered hot, with the size of the plant and the efficiency of operation.

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IXDUSTRIAL A,VD ENGINEERISG CHEMISTRY

I n consideration of the initial cost of investment and the present undeveloped state of the market for this acid and its salts, it is impossible to fix even a tentative price for this product. It is felt, however, that with the establishment of a finished process and the stabilization of demand on a reasonable scale, gluconic acid and calcium gluconate may be produced a t a price comparable with that of citric acid. The writers realize that there are still several important factors t o be worked out if the process is to be established on an efficient industrial basis. Among these are large-scale sterilization of a plant in which an open-pan process is employed, large-scale inoculation of the pans, and large-scale sterilization of the sugar solutions and their distribution to the fermentation pans without contamination. ’These problems are clearly recognized and acknowledged, but it is beyond the scope of the work of this division to investigate them. They are by no means insoluble. In the hands of it competent

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chemical engineer with a knowledge of industrial biological processes they should be readily and satisfactorily worked out. Bibliography Amelung, 2 . physiol. Chem., 166, 161 (1927). Bernhauer, Biochem. Z., 153, 517 (1924). Bernhauer, I b i d . , 197, 278, 287 (1928). Butkewitsch, Ibid., 164, 177 (1924); 182, 99 (1927). Falck and Kapur, Ber., 67, 920 (1924). Herrick and May, J . B i d . Chem., 77, IS5 (1928); U. S. Patent 1,726,067 (1929). Herzfeld and Lenart, Z . Ver. deut. Zucker-Znd., 69, 122 (1919). Ling and Nanji, J . SOL.Chem. I n d . , 41, 28T (1922). hlay, Herrick, Thom, and Church, J . Biol. Chem., 75, 417 (1927). Molliard, CompL. rend., 174, 881 (1922); 178, 41 (1924). Stoll, U. S. Patent 1,648,368 (1927). Stoll and Kussmaul, U. S. Patent 1,703,755 (1929). Takahashi and Asai. Proc. I m p . Acad. Japan, 3, NO.2, 85 (1927). Wehmer, Biochem. Z., 191, 418 (1928).

Line Coordinate Charts for Representing Chemical Engineering Data‘ Edw. A. RavenscroftZ DEPARTMENT O F CHEMICAL

MICHIGAN, AXN ARBOR,

MICH.

The purpose of this paper is to show how line coIn brief, then, any straight HE system of line coordinates may profitably be employed by the chemical line in the Cartesian system ordinates is probably as old as or older than engineer. The principle of line cogrdinates is exrepresents a linear equation the more familiar Cartesian plained, and the relative merits of charts constructed b e t w e e n x and y, and any coordinates, I n general, line in line coordinates and in Cartesian coordinates are point on this line represents coordinates are not so condiscussed. Five examples of line coordinate charts are a solution to the equation. venient or clear as Cartesian given, together with their method of construction and In the line coordinate system coordinates for graphically their use. any point represents a linear reprejenting the relation beequation between x and y, tween two variables; but in some cases they do present dis- and any straight line drawn through this point represents tinct advantages. a solution to the equation. It is clear, then, that a series of straight lines in Cartesian coordinates reduces to a series of Comparison of Cartesian and Line Coihdinates points when transformed into line coordinates. It may be Plane Cartesian coordinate axes consist of two perpendicu- further shown that a series of lines passing through a comlar lines. Any corresponding set of values for two variables, mon point of intersection in Cartesian coordinates reduces to say x and y, is represented by a point so located that its a series of points all lying along a straight line in line coprojection on the x axis reads x, and its projection on the ordinates. If the straight lines in the Cartesian system are y axis reads y. Figure 1 is such a system. On it is plotted all parallel (infinite intersection), then the points to which X, a linear relation between 2 and y-namely, 29 - x = 2. they reduce in line coordinates will all lie on a line parallel Line coordinate axes consist of two parallel straight lines. to the coordinate axes. Any corresponding set of values for the two variables z and If the relation between x and y is empirical and does not y is represented by a transverse straight line intersecting yield a straight line in Cartesian coordinates but rather a the x axis at x and the y axis a t y, as shown in Figure 2 . It smooth curve of some sort, in transferring to line coordinates is this line that is the basis of the name “line coordinates.” the coordinate lines representing corresponding values of On it is plotted the point X, representing the linear rela- x and y will be found to form in general an envelope of tantion 2y - z = 2. It can be proved by analytical geometry gents to some transverse curved line. Corresponding values that any straight line drawn through X will intersect the x and of x and y are read by drawing a straight-line tangent to y axes a t values of x and y which will satisfy the above equa- the envelope and reading x and y a t its intersections with the tion ( 3 ) . coordinate axes. This is the basis of tangential coordinates, I n Figure 1 there is located a point P, the coordinates of of which line coordinates are a special branch. Representwhich are (2, y). Since this point lies on A, its coordinates ing a non-linear relation between x and y by this method represent vnlues of x and y that satisfy the equation repre- is not so convenient as the customary curve in Cartesian sented by A. I n Figure 2 there is drawn a straight line P , coordinates. There is no simple relation between the Cartesian the coordinates of whirh are (2, y). Since this line passes curve and the envelope in line coordinates. There are other through X, its coordinates represent values of x and y that relations between these two coordinate systems, but they lie satisfy the equation represented by the point X. Some- bpyond the scope of this paper (1,3,10). times the name “point equations” is used in place of “line It may be added that corresponding scales are used in the coordinates” because of this fact. two systems. Thus, if the relation between x and y can be represented as a straight line by plotting log x against y 1 Received May 27, 1929. 2 Present address, 677 Valley Road, Glencoe, Ill. in Cartesian coordinates, the straight line may be condensed

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E N G I X E E R I N G , b-XIVERSITY O F