I Case History
OTTO HROMATKA, 1. Chemisches lnstitut der Universitat, Vienna, Austria HEINRICH EBNER, Laboratorium d. H. Enenkel OHG., Linz/Donau, Austria
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Vinegar by Submerged Oxidative Fermentation S u B m R G m OXmATIm fermentation for vinegar production, first reported in 1949, has developed rapidly (7, 2, 4-6, 8-70, 13-27, 23, 30-32). B~ 1957, 80 Acetators ( 7 7, 72), having a total capacity equal to about 30% of the white vinegar produced in the United States during 1945 ( 3 ) ,had been delivered to various parts of the world. Acetators are built in four standard sizes which convert 75, 150, 300, or 600 liters of absolute alcohol in 24 hours. T h e tank is of either stainless steel or wood and all fittings are of stainless steel or plastic. T o start fermentation, the Acetator is filled with a mixture of vinegar, mash, and if necessary, a nutritive substance. Usually, heat generated by the aerating device is sufficient to warm the contents slowly to fermentation temperature (30' C.) which is maintained by an automatic cooling system. Increased acidity is generally titratable within 3 to 10 days. Natural vinegar bacteria should be used for all fermentations except white vinegar for which a special strain cultured in these laboratories is available, as well as a special nutritive substance, Frings Acetozym (2 kg. per liter of mash). The higher the total concentration of alcohol and acid, the better the yield and the less danger of contamination. Fermentations should never be started below 4% acidity and preferably between 6 and 7%. Vinegars containing 12 weight yoof acetic acid can be produced a t full fermentation rate. When acidity begins to increase, fermentation rate grows logarithmically as the number of bacteria multiply. The time needed to double the rate depends on the concentration of alcohol and acetic acid in the mash. For 5% vinegar it is about 3 hours and for 12% vinegar about 10 hours. Fermentation continues until nearly all the alcohol is converted to acetic acid. Then without interrupting aeration, the Acetator is half emptied and the remaining liquid inoculates the next batch. However, because the bacteria are sensitive to environmental change, the fresh mash must be added carefully -it has a high alcoholic content and the fermenting vinegar is highly acid. Thus, a new lag in fermentation is avoided by introducing fresh mash into the impeller of the aerator through an automatic charger, and oxidation continues immediately a t about 80 to 90% of the
theoretical rate as calculated by the full rate of the last cycle and the amount of inoculum and new mash. Logarithmic phase of growth also starts and continues until growth is limited by the quantity of air bubbling through the liquid. Acidification rate increases during growth phase and stays constant a t a maximum value until the end of the cycle. Duration of the following run can be calculated and varied within certain limits by slightly modifying the amount of mash.
Raw Materials Some wines with a high concentration of alcohol, and blends of wine with alcohol, require the addition of 0.01 to 0.04% of ammonium phosphate. If sulfur dioxide has been used as a preservative, it has to be removed with hydrogen peroxide. A special advantage is that cheap wines containing 4 to 6 volume yo of alcohol (vinello, petit vin) can be used. Converting cider to cider vinegar is always difficult in shavings generators
because of sliming. I n Acetators, however, this can be done easily, provided 0.01 to 0.05% of ammonium phosphate is added. Fruit wine containing 4 to 6 volume % of alcohol or if permitted by food laws, even fortified with alcohol u p to 12% can be used. Also, for converting malt mash to malt vinegar, the Acetator offers advantages-birch twigs do not slime, higher yields are obtained, and adding nutritive substances is not necessary. Using a pure alcoholic fermentation when preparing the mash and taking precautions against contamination may further improve quality and yield
(24. Mashes having a high alcoholic content, obtained from raw sugar solutions, can be converted provided 0.05% of ammonium phosphate and 0.05y0 of potassium sulfate is added to the mash. Wine made from sugar cane juice gives excellent vinegar. Also, mashes from fruits such as pineapples, bananas, citrus fruits, raisins, or their wastes can be used, whereas in shavings generators these materials cause sliming. Operation Requirements T o determine when fermentations are ready for discharge, two or three acid titrations and one alcohol determination are usually needed for each run. However, for large amounts of mash having a uniform composition, duration of the run and composition of the vinegar d o not vary because the fermentation process is uniform. Here, only one or two acid titrations per run are needed, together with an occasional check of alcoholic content every three or four runs. For mash containing 8 to 12 volume yo of alcohol a run takes 48 hours, and one having a lower alcoholic content about 24 hours. Power Consumption
The Acetator. Tank is filled with a charging pump, m, and discharged through k. The liquid is aerated with device g (28) which sucks air through a and rotameter h without compressor. Temperature is automatically controlled by thermostat i which opens and shuts the electric water valve, f. A mechanical defoamer, b, breaks froth and exhaust gases leave through tube c
For all vinegars, the Acetator consumes 0.30 to 0.35 kw. hour per liter of absolute alcohol converted. This is more power than shavings generators use; however, power cost is only about 0.5 to 2.0% of that for raw materials, a t least where good quality material is used. Also, higher yields offset this higher power cost. I n making antibiotics by submerged fermentation, a n aeration efficiency, E, (diffused oxygen in liters per watt hour) has been derived (7). I n fermenting VOL. 51, NO. IO
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OCTOBER 1959
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units \\.itti a capacity u p [ o 12.000 liters this t-ffkiency was bt:t\vreii 0.iO and 0.45. 'I tic. Acetator. 'I-ype 600. requires a b o u t 8 i cubic meters o f air per hour. \\.it11 a normal filling hright of 24,000 liters. and consumes 8.5 k~v.-hr. per hour. L'sing this quantity of air and mash (liquid height 3.4 rnrtrrs) \vith an Enrnkel Aerator (27). t h r constant incrrasc in acidity is 0 . 1;?:. prr hour \\.hen gro\vth phase is completed : thus. 36 kg. of acetic acid is produced prr hour. Throrrtically, this requires 19.2 kg. of osygrn ; however. 85 cubic meters of air contains about 23.4 kg. 'I'hrrefore. the surplus oxygen factor is 2.3.4 19.2 = 1.22 Ichich means that 185; of thr o n g e n leaves the generator with the rshaust gases and 82% is consumrd. However, on the avrragc, .4crtators consume only about 7 duced oxygen. Thrrrforr. the aeration vffrirncy for the Acrtatur. Type 600. is 7.0 ' ; ~E. 17,800 I. O ? / h r . X . 8500 watts 1.4- 1. 0 ~
2
watt hr.
a respiration cluoiirnt o f QO, = --4L'sirig 0 cc. of ox)-gen per grani dry \\.right /
of bacteria per hour ( 7 4 . and for each crll, an average size of 7 X 10- I' cc., a dry \\.right of 30c7, and a sprcific \\eight of 1 . the number of bactcria in 1 liter of fc.rnirnring vinegar mash is 3.4 X loL' a i an acidification rate ur n . 1 . 5 ~ ~ per hour. At full capacit). rht: Acetator 600 contains about 8 X hactrria. Cooling- Wa ter Con su mpti on \Vhen 1 liter of absolute alcohol is oxidized to acetic acid, about 2019 kcal. of heat is released. Temperature of the \vater leaving thr cooler is practically constant at 29' C . ; therefore, the amount of water needed to rernove this heat depends chiefly on thr temperature of \cater entering the coolrr. For example: if this temperaturr is 12' C., 170 liters of 'ivater is nerded to remove the heat generated when 1 litrr of alcohol is oxidized. For tropical cliniates, the Acetator is delivered with a n enlarged cooler. I n extreme caws. ho\vever, fermentation temperature can be increased without loss in yield because of efficient aeration-e.g., a t 34' ('.. loss in yield can be only 1 to 2:;;. Yield 'The greatest advdntdge of the Acetator is improved yield. 'Theoretically. 1 liter of absolute alcohol gives 1.036 kg. of acetic acid and 313 grams of water. T h e theoretical increase in volume caused by water during fermentation can be observed in the Acetator. For example. 1000 liters of mash containing 10.0 volume % of alcohol theoretically produces 1020 liters of vinegar containing 103.6 kg. of acetic arid. Therefore, maximum concentration of vinegar is
1 280
theoretically 10%;1. 'I he assumption that 1 volume % of alcohol produces 1 weight % of acetic acid as the basis for calculating yield in the Acetator seems justified. If the percentage of alcohol plus that of acetic acid (total concentration or C K ) remains constant, no losses occur during production. Therefore. the ratio is: GK in vinegar ~... X 100 GK in mash
=
GI; yield
'The acid yield.
x
100
is therrfore al\va)-s lo\vrr than thc. G K yield. Overosidation of acetic acid generally does not occur during initial aeration \\.ithout alcohol. 'Phr point a t which this occurs deprrids on total concentration. Fermrntation can procerd until the alcoholic content is nrarly zero. .Accuracy of the analytical iriethod for determining alcoholic content determines Lvhether the vinegar should be discharged a t 0.30 or O.lO(;& alcohol. Discharging time should tie lrss than a half hour. Hobvever. thr difference between acid yield and G K yield is not a loss because during storage parr of the residual alcohol is converted to acetic acid and part to esters \vhich produce the aroma substances. 'I'his residual alcohol is responsible for the typiral l~ouqurt o f \cine vinegar. \Vhen comparrd ~ i t calculated h yields .icetator yields average higher for cidrr. malt. and \vine vinrgar. and about 5C;; highrr for Ivhite vinegar. For examplr, in a n Acetator bvhich converts 300 liters of absolute alcohol in 24 hours. 184.800 liters of alcohol mash with a n average contrnt of 1.1735. acid and 10.8270,; of alcohol were frrmented continuously for 1469 hours. T h e resulting white vinegar had a n average content of 11.3657; acid and 0.2985; of alcohol. Capacity \cas 326.8 liters of alcohol per 24 hours. GI; )-ield \vas 97.19yL, and acid yield \vas 94.147c. Similar yields are obtained for \cine mashes, and wine mashes fortifird \cith alcohol. Filtration T h e cloudiness in vinegars made by submerged fermentation is caused bv suspended bacteria and insoluble substances from the mash. Unlike operation with shavings generators. the mash need not be fined and filtered before use. Also soluble substances from the mash may slowly precipitate after conversion : however this also occurs in shavings generators and depends on the raw material. With a flotation filter filled with kieselguhr, any vinegar made by submerged fermentation can be filtered
INDUSTRIAL AND ENGINEERING CHEMISTRY
clear. If a suitat)le filwr is not availablc or if clouding is s h i v to clrar. coagulating icith a fining agent. Ivhich must b r found by tests. is nrcrssary. Ho\rever. the fined vinegar milst he filterrd. Sell-clarification by sedimentation d r pcnds on acidity-- r.6.. cider v i n q a r o f lo\< acidity does not clear even after long storage; hut u s u a l l ~within months \vine vinrgar or ivhitr vinegar of high acidity is practical1)- clear. A clrar-filwrrd, submerged vinegar brha\.rs during storagr and bottling like vinrgars made by older methods. But after ii ccrtain prriod flavor and aroma arr hrtter-~-less aroma-producing substances are volatilized and contamination by othrr bacteria is rrdured. References (1) Bryrr, O . , Szeszijar 4 , I65 (1956j. ( 2 ) Bonnat, K., Bull. / p r h . de la linaiqrerit-. S o . 6,
139 (1956).
1.3) BoruR, (:. S., Van I.anen, .J. Sf.? . Ciiew. 39, 934 (1947). ( 4 ) Bourgeois FrPres Pr CXe, S . A , , French Patrnt 1,132,093 (March 5, 195;1. l,5) British Vinegars I>td., Brit. t'atf.nt 727,039 (March 30, 1955 I . ( 6 ) Zbid., 781,584 ( A u ~ 21, . 1957). I(-! Chain, E. B., Gualandi, G., Rend. i \ 1 . super. sanzlh 17, 5 (1954j. 1.81 Dothey, G., Belg. Patrnt 509,550
(Marrh 15, 19.52). €rings, H., Brit. Patrnt 731,804 (June
(91
15, 1955). ( 1 0 1 Frings, H., (krrnan Patrnt 898,134
(Oct. 22, 19531. ( 1 1 1 €rings, H.,
Intern. I
