June, rgzo
T H E JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
should be very few OH ions. Excess sodium bicarb o n a t e does not interfere. I n t h e presence of sufficient hydrochloric a d d t h e reaction goes from right t o left. It is quantitative in both directions. Salts of copper interfere in t h e above determination of t h e arsenic form of arsenic, b u t i n most cases t h e copper content will be low enough t o be negligible. Should. copper be found in a n appreciable quantity, however, i t should be determined a n d a correction applied t o t h e thiosulfate titration, remembering t h a t e a c h mole of copper will liberate one mole of iodine from potassium iodide. If copper is present in a n y
quantity t h e a m o u n t of potassium iodide added should be increased accordingly. T h e only other constituents likely t o interfere with t h e determination of the arsenic form have been removed by t h e method of procedure. Nitrous a n d nitric acids would be volatilized during t h e heating. Ferric iron would be precipitated by t h e sodium carbonate a n d removed in the subsequent filtration. This method has been checked against various other arsenic methods a n d results h a v e been found t o agree very well. Duplicate determinations of t h e same acid have been found t o check closely.
LABORATORY AND PLANT SEED-CULTURE METHODS IN THE PRODUCTION O F ACETONE AND BUTYL ALCOHOL BY A FERMENTATION PROCESS
By Horace B. Speakman DEPARTMENT OF
ZYMOLOGY,
TORONTO U N I V E R S I T Y , TORONTO, CANADA
Received January 15, 1920
A bacteriological fermentation process may be divided for descriptive purposes into t w o parts: firstly, t h e preparation, fermentation, a n d distillation of t h e large bulk of raw material; and secondly, t h e preparation, i n a n active a n d pure condition, of t h e necessary volume of culture, t o act as seed-culture for t h e final fermentation. I n a previous article' a n outline was given of t h e essential features of a plant concerned in t h e first of these divisions of t h e process. I n t h e present communication it is proposed t o consider the a p p a r a t u s a n d methods of working in t h e production of seed. Emphasis must be laid i n t h e first place o n the necessity of a n adequately equipped laboratory. Furthermore, t h e principles of bacteriology must be allowed t o control a n d direct not only t h e laboratory, b u t t h e operations of t h e entire plant. T h e problem consisted in finding t h e best method of developing from a laboratory t u b e culture a sufficiently large a n d active volume of culture t o s t a r t a fermentation in a large fermenter, containing 24,000 gal. of mash, which will occupy t h e minimum number of hours a n d give t h e maximum yield of products. Such questions as t h e following a t once arose: ( I ) W h a t is t h e correct ratio between t h e volumes of t h e seed-culture a n d t h e mash t o be inoculated? ( 2 ) At what stage in t h e fermentation must t h e cult u r e be used as seed? ( 3 ) C a n t h e organism growing in maize mash be subcultured indefinitely i n t h e same t y p e of medium without loss of vitality? If not, what is t h e number of generations between t h e tube a n d t h e fermenter which will insure t h e maximum efficiency in t h e 1a t t er 7 Because t h e great demand for acetone made impossible small-scale, preliminary experiments, these ques1
Speakman, J . Sac. Chem. I n d . , 38 (1919), 155.
tions were answered in t h e factory rather t h a n in t h e laboratory. T h e seed plant was often modified, and, when t h e armistice was signed, i t represented t h e s u m of t h e experience gained during t h e previous two a n d a half years. T h e second question will be considered i n detail. During t h e fermentation t h e acidity of t h e mash rises steadily for a b o u t 1 5 hrs. a n d then falls for about t h e same number of hours. I n all stages of t h e seed process t h e culture was transferred t o t h e next b a t c h when t h e acidity h a d just begun t o fall, for t h e following reasons: At this point t h e organism had ceased dividing a n d had passed through t h e chain stage. T h e medium, however, had not yet separated into a clear fluid with a slimy floating head which contained t h e majority of t h e bacteria, b u t was still homogeneous a n d contained t h e maximum number of free motile organisms. I n this condition t h e culture could be divided equitably between several batches of mash. I t was also found t h a t when contamination occurred only one species of foreign organism developed, a n d this type was responsible for t h e stoppage of t h e fermentation. I n t h e early stages t h e contamination could not be detected by its morphology or b y chemical tests, b u t toward t h e end of the fermentation t h e contamination stood out clearly i n faintly stained preparations, a n d t h e acidity of t h e mash did not fall b u t rose t o a very abnormal maximum. For these reasons i t was considered advisable t o wait until t h e acidity had commenced t o fall, a n d until slides had been examined very carefully, before passing t h e culture into a larger volume of mash. A further question arises i n connection with t h e number of vessels t o be included in t h e system. Supposing t h a t 1,000 gal. of culture are required each d a y for four large fermenters, is i t advisable t o prepare this i n one or more vessels? If one vessel is used there is a saving i n time a n d labor, and, what is of greater importance, b y reducing t h e number of pieces of apparatus t o t h e minimum t h e danger of contamination is correspondingly reduced. On t h e other hand, if contamination does occur, t h e plant m a y be idle for a day. Furthermore, whilein general it is possible t o arrange t h a t t h e culture be i n t h e right condition when required, occa-
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sional delays may occur or t h e fermentation may be unusually rapid. T h e operator must then divide among four fermenters the 1,000 gal. of culture which have separated into clear beer and head. Not only is this almost impossible, b u t often the head refuses t o pass out of t h e seed vessel along t h e lines, a n d the fermentations are slow in t h e fermenters. As far as possible, therefore, the culture for each final fermentation represented a complete smaller-volume fermentation. MECHANICAL DETAILS OF THE P L A N T
MATERIAL-The seed plant as a whole was composed of copper vessels. I n many cases i t was found t h a t the first fermentation in a new vessel was slow a n d incomplete, and upon examination of the interior t h e bright metallic surface was found tarnished b y thin
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completely immersed in baths of dilute carbolic acid. STERILITY DEVICES-certain mechanical devices to preserve sterility were used on all t h e vessels. T h e drains from t h e lines were of the type previously described. T o provide for agitation of t h e mash during t h e various operations a central shaft passed through a gland in t h e domed roof a n d was supported b y a step bearing on the bottom of t h e vessel. T o prevent contamination through t h e gland a cup was brazed on t h e top of t h e latter (Fig. I). During t h e cooking a n d cooling periods this cup was filled with a mobile mixture of grease, butyl alcohol, a n d carbolic acid, which worked down into t h e gland a n d acted as a lubricant, while the excess in the cup indicated whether t h e gland was leaking when the vessel was under pressure. I n this indirect way, and by preventing t o some exkent t h e passage of air into the vessel ,during t h e cooling period, t h e cup helped t o eliminate this /I source of contamination. Another fruitful source of trouble was the gauge glass. Those originally placed o n t h e fermenters were removed, b u t the gauge glass was a n essential part of the equipment of t h e smaller vessels. I n order t o increase their usefulness a n d FIG.Z-GAUGE GLASSAND SAMPLING COCK lessen t h e danger, large brass mountings were designed for all t h e vessels. Instead of t h e usual narrow bore of steam mountings, one which was half a n inch in diameter was called for, t o prevent the closing u p of t h e valves with mash. If this did occur t h e glass a n d fittings could be cleaned out with steam. Owing t o t h e structure of t h e gaugeglass valves there is always a danger t h a t leaks will develop around t h e spindle, a n d they were accordingly assumed t o be always in this condition. A low-pressure steam connection (Fig. 2 ) was made in t h e upper valve. During t h e cooking a n d fermentation periods t h e valves were open a n d t h e steam connection valve was closed. T h e t a n k was under steam or gas pressure during these periods. During t h e cooling period a n d early hours of the fermentation, t h e t o p valve was closed a n d a slight amount of steam was turned on. After t h e condensed steam in t h e line washed out t h e glass, t h e bottom valve was closed, steam still being turned on. The gauge glass gradually filled with sterile water under a slight pressure and the passage
i
-4
i
FIG.I-ANTISEPTIC OIL CUP
deposits of some unknown, dark substance. If this 'thin layer were removed a n d a new surface of metal exposed, the same result was obtained when t h e t a n k was used again. T h e vessel should therefore be washed out b u t not scoured. It is suggested t h a t t h e thin deposit over t h e copper reduces the action of t h e fermentation acids on t h e metal, a n d thus diminishes t h e formation of toxic metallic salts. Aluminum vessels have been used with success in Englana, but from t h e bacteriological standpoint they offer no advantages as compared with copper vessels, a n d from t h e mechanical standpoint they are much inferior. sIruATIo~-The vessels should be so arranged in relation t o one another, t h a t t h e culture can flow by gravity with fair rapidity from one stage t o t h e next. This arrangement obviates t h e use of pumps in the seed lines, which is a great help in the preservation of sterility. If pumps are required i t is advisable t o use the rotary type a n d they should be
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June,
1920
T H E J O U R N A L O F I N D U S T R I A L A N D E N G I N E E R I N G CIf E J f I S T R Y
of air into the tanli through t h e gaugs valves was prevented. A sampling cock
in
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cool, dry place after the fcriiicntatiuii censed.
slight deposits remained on below the plug. These deposits contai
dcpended upon. A t the close of the cookin sample of the mash >was examined under scope. If organisms mere t o he seen in t h
was
with tlle sterility test the following steps were taken: I n the il,teri,n between tile enlptpinR and filling of t h e vessel, a slight amount of steam was kept passing into it. The valves and coc~c ere partially closed and a t intervals they were all closed for a few minutes. When pressure developed ala, J-sauuL,No COCk tile valves were opelied in turn and the steani allowed t o rush through them to thoroughly clean the body of the valve. When the t a n k was once more full the cock was cleaned out with cottonwool soaked in dilute carbolic acid, and plugged with a clean piece of cotton. In Fig. 3 are given the details of a sampling cock which was tried with success on one of the seed-vessels a t the close of 1918. The main portion of the apparat u s was an ordinary one-half inch cross-valve, the whole body of which could be thoroughly cleaned and sterilized with steam. During the cooking period and between the taking of samples the whole of t h e valve on the discharge side was full of condensed steam under pressure. The small cock was cut down below the r~lu~?. . I As an estra urecnution the cock was immersed in a cup of antiseptic, suspended from the valve ahovc. This valve should eliminate sources o f contamination, causes of delay, and also tedious operations. L A N O X A T O R Y C U L T U K K JICT€IOD
T h e starting point in the preparation of the seed was a test tube containing the products of 3 fermentation, maize residues, and spores of t h e haciiius (Pig. 4). The t u b e had been stored for 3 mo.
the develo
desiccator wns not used. If the inoculnnt was placed at the base of the starch gel and the tube was not shaken, bubbles of gas vcry soon surrounded tire culture and gradually created tliroughout the whole tube an anaerobic atmosphere. The success of this operation depended on the vitality of the bacillus, and more specialized technique must be adopted when sluggish cultures or spores are used. On the following day two of the tnhes were used t o inoculate tu.0 goo-cc. Erlenmeyer Rasks containing 300 cc. of 5 per cent mash (Fig. 4 ) . The remaining tube cultures %ere incubated for two more days and, if necessary, Were then used t o maintain the stock of spore tubes.
Fro
4-1,anoxnl'oxu
Cl:i.r"KEs
Smears of the cultures were made, converted into i,erniaiient preparations, nnd labelcd for future reference. A n important feature of the system as t h a t a new culture was start.ed each day and became the starting point for t h e whole of a l a t a day's factory opcrations. The minimum amount of subculturing was done i n the laboratory, and consequently the
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danger of slow fermentations in t h e factory was reduced to a It was found ilnpossible to predict from the inorphological characteristics of the organism i n t h e early laboratory cultures what t h e findfermentation be therefore, the number of generations between the spore culture and t h e fermenter must be reduced t o thk minimum. It is believed t h a t i t was largely due t o the adoption of this system t h a t the time of the final fermentation period was reduced in z years from an average of 48 hrs. t o a n average of 3 0 hrs. ~
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No. 6
STIRRING
