The precipitated calcium succinate was filtered at 39? C. by vacuum filtration. Broths from 1L and 2 L fermentations were filtered using Whatman No. 1 filter paper (Whatman Inc., Clifton Heights, N.J.) in a 17 cm dia. ceramic Buchner funnel. The filtrate was collected in a 2 L Pyrex vacuum flask. The filter cake was washed with the minimum volume required to remove all the filtrate. For larger fermentations a 20-ounce cotton twill filter cloth in a 12-inch diameter Buchner funnel was used for the filtration. The filtrate was then heated to 80? C., seeded with calcium succinate and mixed for 15 minutes allowing equilibrium to be established. The hot slurry was then filtered. The calcium succinate filter cake was washed with enough 80? C. water to remove all filtrate.

Conversion to Succinic Acid

The washed calcium succinate cake was reslurried to 33% w/v solids with water. Concentrated sulfuric acid was slowly added, in 10% excess of the amount required to convert all of the calcium succinate to succinic acid(cas:110-15-6). This slurry was mixed well and allowed to react completely. Then the resulting mixture of calcium sulfate (gypsum) solids and succinic acid(cas:110-15-6) solution was heated to 80? C. and vacuum filtered through 20-ounce cotton twill filter cloth. The filter cake was washed with enough 80? C. water to remove all the succinic acid.

Purification of the Succinic Acid(cas:110-15-6)

After calcium sulfate (gypsum) filtration, residual cations, anions, and nitrogenous materials still remained in the succinic acid(cas:110-15-6) filtrate. A strongly acidic cation exchange resin, in the acid form, such as Dowex 50WX8 (Dow Chemical Co., Midland, Mich.), was used to remove the calcium and other cation impurities. The solution was then treated with a weakly basic anion exchange resin in the free base form, such as Amberlite IRA-94 (Rohm & Haas Co., Philadelphia, Pa.), which removes sulfate and other strong anionic contaminants without removing the succinic acid.

Two-inch diameter glass columns were used for ion-exchange purification of the succinic acid(cas:110-15-6) stream. The succinic acid stream was treated continuously in the columns at flow rates of 5-10 bed volumes per hour. Columns were regenerated using dilute (1-2M) sodium hydroxide or hydrochloric acid, as appropriate.

Analytical Methods

The organic acid fermentation products were determined using high-performance liquid chromatography (HPLC) as described by Guerrant et al., J. Clinical Microbiol., v. 16 (2), 355 (1982). Components were analyzed chromatographically by elution with 0.006 N H2S04 from a cation-exchange resin in the hydrogen form. A Waters Model 60 HPLC system with a Bio-Rad HPX-87H column and a Waters Model 410 Refractive Index detector were used in this analysis. The carbohydrate was determined by a YSI dextrose analyzer (Yellow Springs Instrument Company, Yellow Springs, Ohio). All the results were reported as grams per liter.

Total Kjeldahl nitrogen (TKN) was determined using a Tectator Model 1030 Kjeldahl system and protein was calculated by multiplying %TKN by 6.25. Calcium was determined spectrophotometrically as per Sigma Chemical Co. test kit #587-A using Bausch & Lomb Spectronic-20 Spectrophotometer (Milton-Roy Co., Rochester, N.J.). Sulfate determinations were made gravimetrically by the barium precipitation method of Fronson, M. A., ed., Standard Methods (16th), Amer. Chem. Soc., 464 (1985).