# Patent

## Title
Shaft furnace for thermolytic distillation of hydrocarbon fuel

## Abstract

This is a division of application Ser. No. 504,598 filed Sept. 9, l974, now U.S. Pat. No. 3,939,139.

## Background
There is provided, according to the present invention, an antibiotic substance effective in inhibiting the growth of Gram-positive bacteria which is of the Formula I. Chemically, this substance is known as 1-(S)-hydroxy-2-(S,S)-valylamido-cyclobutane-1-acetic acid.

There is further provided according to the present invention, a process for the production of such antibiotic substance of the Formula I which comprises cultivation of a strain of Streptomyces sp, X-1092 in an aqueous carbohydrate solution containing a nitrogenous nutrient under submerged aerobic conditions until substantial activity versus gram-positive bacteria is imparted to said solution and then recovering said compound of the Formula I from said solution.

The organism producing the antibiotic of the present invention is a new species designated Streptomyces sp. X-1092. A culture of the living organism, given the laboratory designation X-1092, has been deposited in the U.S. Department of Agriculture, Agriculture Research Service, NRRL, Peoria, Ill., and added to its permanent collection of microorganisms as NRRL 8047.

The representative strain of Streptomyces sp. X-1092 has the following characteristics.

On solid media, this organism grows in a fashion that is characteristic for members of the genus Streptomyces: abundant mycelial growth penetrates into the agar, and the aerial mycelium breaks up in characteristic chains of spores while there is no fragmentation of the immersed mycelium. The aerial sporulation system belongs to the type retinaculum-apertum (RA), with loose spirals, loops and hooks of small diameter.

The formulation of the media used in the description of the growth characteristics, is described below.

Medium 1: "Fermentation medium": Difco Bacto Thermoactinomyces fermentation medium to which 1.5% agar was added.

Medium 2: BBL(Baltimore Biological Laboratory) Czapek-Dox, to which 1.5% agar was added.

Medium 3: Difco Bacto potato dextrose agar.

Medium 4: "Y+M medium": yeast extract, 0.4%; malt extract, 1.0%; dextrose, 0.4%; agar, 2%; pH 7.3.

Medium 5: "Oatmeal-glucose medium": Gerber's oatmeal, 5%; dextrose, 2%; agar 2% in tap water; pH 7.0.

Medium 6: "Tomato paste medium": dextrose, 1% K.sub.2 HPO.sub.4, 0.1%; tomato paste, 2%; Wilson's Medopeptone, 0.1%; CaCO.sub.3 ;0.2%; agar, 1.5% in tap water; pH 6.8-7.3.

Medium 7: "Tomato-soy medium": same as medium 6, to which 1% soyalose (Central Soya Co.) has been added.

Medium 8: "Pablum medium": 6% Pablum mixed cereal in cheesecloth bag dipped in and out of boiling tap water for 2 or 3 minutes. The water lost by evaporation is replaced, and agar is added to 1.5%.

Medium 9: "Tomato-oatmeal" medium": baby oatmeal (Gerber's), 2%; tomato paste, 2%; agar, 2%, in tap water; pH 6.8-7.3.

Medium 10: "Yeast extract medium": yeast extract, 1%; dextrose, 1%; agar, 1.5%, in tap water; pH 6.8.

Medium 11: "Glucose-asparagine medium": dextrose, 1%; asparagine, 0.05%; K.sub.2 HPO.sub.4, 0.05%; agar, 1.5%; pH 6.8.

Medium 12: "Glycerol-asparagine medium": glycerol, 1%; asparagine, 0.1% K.sub.2 HPO.sub.4, 0.1%; agar, 2% in tap water; pH 7.0.

Medium 13: "Starch-casein medium": soluble starch, 1%; casein, 0.1%; K.sub.2 HPO.sub.4, 0.05%; MgSO.sub.4, 0.05%; agar, 1.5%; pH 7.4.

Medium 14: "Emerson's medium": beef extract, 0.4%; peptone, 0.4%; Nacl, 0.25%; yeast extract, 0.1%; dextrose, 1%; agar, 2%; pH 7.0.

Medium 15: "Bennett's medium": yeast extract, 0.1%; beef extract, 0.1%; N-Z-Amine A (casein hydrolysate from Sheffield Inc.) 0.2%; dextrose, 1%; agar, 1.8%; pH 7.3.

Medium 16: "Amidex medium": Amidex (Corn Products Co., Decatur, Ill.) 1%; N-Z-Amine A, 0.2%; beef extract, 0.1%; yeast extract, 0.1%; CaCl.sub.2, 2H.sub.2 O, 0.0014%; agar, 2%; pH 7.3.

Medium 17: "Sporulation medium" (ATCC medium number 5): yeast extract, 0.1%; beef extract, 0.1%; tryptose 0.2%; glucose, 1%; FeSO.sub.4, trace; agar, 1.5%; pH 7.2.

Media 18, 19, 20, 21, 22, 23 are, respectively, media 2 through 7 as described by Shirling, E. G., and Gottlieb, D., Methods for characterization of Streptomyces species, International J. of Systematic Bacteroil., 16, 313-340, 1966.

Unless otherwise indicated, the observations reported have been performed after a period of incubation of 14 days at 28.degree. C. The characteristics of growth in these solid media is summarized in the Table below. Colors of upper surface and reverse are named according to Ridgway's Color Standards and Color Nomenclature, Washington, D.C., 1912. Characteristics of Color of upper surface Color of reverse of Medium aerial growth of colonies  colonies __________________________________________________________________________ 1  abundant; wrinkled; pallid quaker drab dirty cream buff good sporulation turning to medium deep center turning to mouse gray toward the dark olive and pale edges; whitish edges olive buff toward the edges 2  fair; flat, thin whitish      whitish sporulation 3  almost none 4  abundant; wrinkled; pale mouse gray dirty olive ocher good sporulation; medium becomes yellow; droplets of yellow exudate on colonies 5  none 6  abundant; wrinkled; light mouse gray, honey yellow center granulated;  with whitish edges and light brownish sporulation thicker       olive toward the at the edges; yellow      edges, with pigment diffuses          mustard yellow into medium               edges 7  abundant; wrinkled; pale mouse gray; off- pale mouse gray with good    white edges  center, mouse sporulation; medium       gray toward the turns light yellow        edges, and off- white edges 8  abundant; wrinkled; pale mouse gray and mustard yellow granulated, good off-white edges center turning sporulation               buffy citrine at edges 9  abundant; wrinkled; light mouse gray with honey yellow center granulated; good off-white edges; later turning light sporulation; traces mouse gray with gray brownish olive of light yellow edges        toward the edges pigmentation in the medium 10  abundant; wrinkled; light mouse gray with antimony yellow, good sporulation; off-white edges which and in parts, antimony-yellow in time become pallid ochraceous tawny exudate; yellow mouse gray diffusible pigment 11  good; wrinkled; barium yellow vegeta- citron yellow scanty sporulation tive mycelium off-white in color 12  abundant; thin cream color vegeta- primuline yellow sporulation, white tive mycelium; later, in color; colonies pallid mouse gray to granular, cracking light mouse gray, with with age; light off-white edges yellow diffusible pigment 13  abundant; light vegetative mycelium barium yellow sporulation; yellow primrose yellow; pigment produced; sporulated areas pallid clearing around the mouse gray with off- colonies (hydrolysis white edges of starch and/or casein) 14  abundant; fairly vegetative mycelium dirty mustard yellow abundant sporulation Naples yellow; spore center turning dirty areas pallid mouse buffy brown and Naples gray turning off- yellow at the edges white at the edges 15  abundant; and good pale mouse gray; edges wax yellow center sporulation, which light mouse gray; off- turning into dirty is thinner at the white edges  olive citrine close center of colonies;       to the edges drops of exudate on colonies; light yellow pigment diffuses into medium 16  good growth and vegetative mycelium mustard yellow center sporulation  chamois; sporulation turning to antimony off-white with mouse yellow toward the gray patches; edges vegetative mycelium at edges of colonies becomes yellow ocher 17  abundant; poor vegetative mycelium olive ocher center sporulation; honey yellow; turning to honey light yellow sporulated areas light yellow toward edges pigment diffusing mouse gray with off- into medium  white edges 18  abundant; wrinkled; mouse gray with few fuscous; paler indenta- with surface cracks; pale gray patches tions immersed into good sporulation;         the agar light yellow diffusible pigment 19  good, flat, with vegetative mycelium cream buff ruffled edges; cream buff; sporulated thin sporulation areas pallid mouse gray 20  abundant; smooth, mouse gray at center deep grayish olive with ruffled edges; and pale mouse gray center changing to good sporulation; near edges; off-white chamois and cream lumps near the edges        buff toward edges center of colonies; very little clear- ing around the colonies 21  abundant; with mouse gray   mouse gray good sporulation 22  fair; no sporulation; Naples yellow mustard yellow light yellow pigment diffuses into the medium __________________________________________________________________________

Unless otherwise indicated, the observations reported have been performed after a period of incubation of 14 days at 28.degree. C. The characteristics of growth in these solid media are summarized in the Table above. Colors of upper surface and reverse are named according to Ridgway's Color Standards and Color Nomenclature, Washington, D.C., 1912.

The species Streptomyces X-1092 described herein includes all strains of streptomyces which form a compound of the Formula I and which cannot be definitely differentiated from the strain NRRL 8047 and its subcultures including mutants and variants. The compound of the Formula I is identified herein and after this identification is known, it is easy to differentiate the strains producing a compound of the Formula I from others.

Streptomyces sp. X-1092, when grown under suitable conditions, produces a compound of the Formula I. A fermentation broth containing Streptomyces sp. X-1092 is prepared by inoculating spores or mycelia of the compound of the Formula I -- producing organism into a suitable medium and then cultivating under aerobic conditions. For the production of a compound of the Formula I, cultivation on a solid medium is possible but for production in large quantities cultivation in a liquid medium is preferable. The temperature of the cultivation may be varied over a wide range, 20.degree.-35.degree. C, within which the organism may grow but a temperature of 26.degree.-30.degree. C and a substantially neutral pH is preferred. In the submerged aerobic fermentation of the organism for the production of a compound of the Formula I, the medium may contain as the source for carbon, a commercially available glyceride oil or a carbohydrate such as glycerol, glucose, maltose, lactose, dextrin, starch, etc. in pure or crude states and as the source of nitrogen, an organic material such as soybean meal, distillers solubles, peanut meal, cotton seed meal, meat extract, peptone, fish meal, yeast extract, corn steep liquor, etc. and when desired inorganic sources of nitrogen such as nitrates and ammonium salts and mineral salts such as ammonium sulfate, magnesium sulfate and the like. It also may contain sodium chloride, potassium chloride, potassium phosphate and the like and buffering agents such as sodium citrate, calcium carbonate or phosphates and trace amounts of heavy metal salts. In aerated submerged culturing procedures, an anti-foam agent such as liquid paraffin, fatty oils or silicone compounds is used. More than one kind of carbon source, nitrogen source or anti-foam source may be used for production of a compound of the Formula I. Generally the cultivation is continued until at least several hundred mcg/ml of a compound of the Formula I has accumulated in the medium.

The following Examples will serve to illustrate this invention without limiting it thereto.

Spores of Streptomyces sp. X-1092, were added to 6-liter Erlenmeyer flasks containing 2 liters of Trypticase soy broth (Baltimore Biological Laboratories). The flasks were incubated at 28.degree. for 76 hours on a rotary shaker [240 rpm with a 2 inch stroke]. Four liters of inoculum was added to 200 liters of fermentation medium containing (in g/liter): K.sub.2 HPO.sub.4, 7.0; KH.sub.2 PO.sub.4, 3.0; (NH.sub.4).sub.2 SO.sub.4, 1.0; sodium citrate, 0.5; MgSO.sub.4. 7H.sub.2 0, 0.1; and D-glucose, 2.0 (autoclaved separately). The culture was incubated at 20.degree. in a 380-liter fermentor, aerated at 113 liters per minute and agitated at 260 rpm. Silicone anti-form (Dow Corning AF) was added as needed to control frothing. After 66 hours of incubation, the fermentation broth was filtered through infusorial earth.

The clarified broth from two 200 liter fermentations was applied to 50 liters Dowex 50WX-4 resin, styrene-divinyl benzene-sulfonic acid ion exchange resin Ca 50- 100 mesh, in the H+ form. After washing with 200 liters distilled water, the resin was eluted with 400 liters 5% aqueous pyridine solution. The eluate which contained 33 g solids was evaporated under reduced pressure to 2 liters, the pH adjusted to 2.5 by addition of 5N HCl and the solution applied to a column (70 cm ht) containing 2.5 liters Bio-Rad AG50WX-4 resin, styrene-divinyl benzene-sulfonic acid ion exchange resin, 100- 200 mesh, in the Na+ form, which had been equilibrated with 0.2M sodium phosphate-citrate buffer, pH 4.2. The resin was then eluted with the same buffer and the activity was obtained at an elution volume of 11-13 liters. This fraction was desalted by readsorption of the antimetabolite onto 1.2 liters Bio-Rad AG50WX- 4 resin, 50- 100 mesh, in the H+ form followed by elution with 10% aqueous pyridine solution. The eluate was evaporated under reduced pressure to a small volume, the concentrate was treated with charcoal, the filtrate from the charcoal step was again evaporated and 1-(S)-hydroxy-2-(S,S)-valylamido-cyclobutane-1-acetic acid was crystallized from ethanol-water (9-1):m.p. 247.degree.-250.degree.; [.alpha.].sub.D.sup.25 + 8.4 (c 1, H.sub.2 O), -31.2 (c 1, 5N HCl); pK.sub.1 3.8, pK.sub.2 7.5; ir (Kbr disk) 3365 and 3240 (OH and amide NH), 1660 and 1520 (sec amide), 1615 and 1390 cm.sup.-.sup.1 (carboxylate); nmr (D.sub.2 O, 20 mg, ext TMS), .delta. 4.61 ##STR1## 4.23 (d,1 J = 6Hz, 2.92 (3.18 with DCl) ##STR2## 2.30-2.80 (m,5), 1.47 (d,6, J = 6.5Hz, ##STR3## Anal. calcd for C.sub.11 H.sub.20 N.sub.2 O.sub.4 : C 54.03, H 8.25 N 11.47. Found: C 54.11, H 8.40, N 11.57.

As indicated above the compound of the Formula I and its salts are active against gram positive bacteria. Listed in Table 1 which follows are the inhibition zone diameters (mm) for several illustrative organisms as determined by agar-diffusion testing. As is apparent from the foregoing properties, the antibiotic of the Formula I is useful for suppressing the growth of gram positive organisms.

The antimicrobial spectrum of 1-(s)-hydroxy-2-(S,S)-valylamido-cyclo-butane-1-acetic acid was measured in a chemically defined medium.sup.a by the paper disc agar-diffusion technique. The results are given in Table 1. The activity was limited to gram positive organism. __________________________________________________________________________ Antimicrobial Spectrum Test Organism      Inhibition zone diameter (mm) __________________________________________________________________________ Bacillus cereus ATCC-6464 60 Bacillus sp. ATCC-27860 25 Bacillus subtilis NRRL-558 34 Streptomyces cellulosae ATCC-3313 34 Micrococcus glutamicus ATCC-13761.sup.b 20 Escherichia coli B  0 Pseudomonas ovalis NRRL-22 0 Candida albicans NRRL-477.sup.b 0 Pullularia pullulans QM-279c 0 __________________________________________________________________________

a. Paper-disc agar-diffusion assays were performed with 12.7 mm discs each containing 12 m.mu.  of a compound of the Formula I in Davis Minimal Agar.

b. Biotin was added to the medium at 100 g liter to insure ample growth of these test organisms.

The compound of the Formula I also evidenced anti-metabolite activity. This activity was determined by counter diffusion methods which have been described in the literature in Journal of Antibiotics, Volume 27, pp. 229-233, 1974. against Streptomyces cellulosae. The activity of the compound of the Formula I was non-competitively reversed by the addition of either L-cysteine or L-cystine to the medium. Partial reversals were observed with either L-methionine or D,L-homocysteine. Other common amino acids, nucleosides and water soluble vitamins did not reverse the activity of a compound of the Formula I. In addition glutathione and dithiothreitol did not reverse the inhibition, so it can be concluded that the reversal by cysteine is not due to chemical inactivation by thiols.

Only slight reversal of inhibition against the three species of Bacilli and Micrococcus glutamicus was observed with L-methionine, L-cystine and L-cysteine; no reversal was observed with other common amino acids mucleosides and/or water soluble vitamins.

As is indicated above, the compound of the Formula I is prepared under submerged aerobic conditions. Preferably submerged fermentation in tanks is used for production of substantial quantities of the X-1092 antibiotic in accordance with conventional procedures. Small quantities of antibiotic are obtained by shake-flask culture. As is customary in aerobic submerged culture processes, sterile air is blown through the culture medium. For efficient growth of the organism and production of the antibiotic of the Formula I, the volume of air employed in the production is above 0.1 volume of air per minute per volume of culture medium. Optimum growth occurs when the volume of air employed is between 0.6 and one volume of air per minute per volume of culture production medium. The production of antibiotics can be followed during the fermentation by testing samples of the broth for antibiotic activity against organisms known to be sensitive to the antibiotic. The bioassay is conveniently effected by paper disc assay on agar plates.

As is the custom, maximum antibiotic production occurs within 2-6 days in either large tank or shake-flask fermentation. Commonly maximum production of antibiotic activity is realized within 5-6 days.

Following its production under submerged aerobic conditions the compound of the Formula I can be recovered from the fermentation broth by methods commonly employed in the fermentation art. The antibiotic activity produced during fermentation of a compound of the Formula I-producing organism occurs in the antibiotic broth. Accordingly, isolation techniques employed in the production of such antibiotics are designed to permit maximum recovery of the antibiotic from the broth. Thus, for example, mycelia and undissolved solids are removed from the fermentation broth by conventional means such as filtration and the antibiotic of the Formula I is recovered from the filtered broth by techniques such as ion exchange or adsorption.

The compound of the Formula I having the basic amino group and the acid COOH group can form salts with both acids and bases. It is easily soluble in alcaline solutions such as aqueous solutions of alkaline metal and alkaline earth metal hydroxides. Thus, solutions of a compound of the Formula I in aqueous sodium hydroxide or calcium hydroxide forms the sodium or calcium salt respectively which can be isolated by conventional procedures. In like manner are formed salts of organic bases such as amine salts and the like. Similarly, the compound of the Formula I can form salts with acidic substances and such substances can be prepared by conventional techniques with such pharmaceutically acceptable acids as hydrochloric acid, hydrobromic acid, sulfuric acid and the like. All that is required of the salt is that it provide a pharmaceutically acceptable salt of a compound of the Formula I.

As is indicated above, compounds of the Formula I and its salts possess the property of adversely affecting the growth of certain Gram-positive bacteria. It is useful in wash solutions for sanitary purposes as in the washing of hands and the cleaning of equipment, floors or furnishings of contaminated rooms or laboratories; It is useful also for suppressing the growth of sensitive organisms in plate assays and other microbiological media.

## Claims

1. The process for the production of an antibiotic substance of the formula ##EQU2## which comprises cultivating a strain of Streptomyces sp. X-1092 deposited as NRRL 8047 in an aqueous carbohydrate solution containing a nitrogenous nutrient under submerged earobic conditions until activity versus Gram-positive bacteria is imparted to said solution and then recovering said antibiotic of the formula I from said solution.