[0001] This application claims the benefit of US Provisional Application No. 61/329,894 filed April 30, 2010, the subject matter of which is hereb incorporated by reference.

Technical Field

[0002] This disclosure relates to processes for the direct production of NH OOC-R- COOH compounds from fermentation broths containing NH/ ^" OC-R-COO ^" H compounds, H/ ^' OC-R-COOH and/or HOOC-R-CO H compound acids. ft. also relates to the conversion of the NH/ OOC-R-COOH compounds so obtained to HOOC-R-COOH compound acids. Background

[0003] Certain carbonaceous, products of fermentations are seen as replacements for petroleum-derived materials for use as feedstocks for the manufecture of carbon-containing chemicals. One category of such products is NH/ ^" OOC-R-COOH compounds- including moftoam ^' tnoniufn fumarate,. monoammonium malonate, monoammonium nialaie, monoammonium giutarate, monoammonium eiiraconate, monoaramonium itaconate, monoammonium mueonate, monoainmonium sebacate and monoamrnoniurn dodeeanedlonaie.

[0004] It would be desirable to have a process for the direct production of such substantially pure NH OOC-R-COOH compounds from NH/ ^{" "} OOC-R-C O ^" NPs/ compounds, NH/ OOC-R-C OH compounds and/or HOOC-R-COOH compound acids in fermentation broth.

Summary

[0005] We provide such a process by economically producing high purity NH4 ⁺ OOC-R- COOH from a clarified NH/ ^" OOC-R-COO ^" NH/-eontainmg; fermentation broth, wherein R may be, but is not limited to, C¾, CH=CH, CH ₂ -CH(OH), (C¾} _3> C ^H, C¾- C=CH _2! CH=€H-CH-C¾ (CH ₂ ) _8. and (CH ₂ )„ We thus provide a process for making Nil/ ^' OOC-R-COOH from a clarified NH/ ^" OOC-R-COO ^* NH/-containing fermentation broth, including (a) distilling the broth to form an overhead that comprises water and ammonia, and a liquid bottoms that comprises N¾ ⁺ OOC-R-COOH, at least some NH OC-R-COO ^" Mi*' ^" , and at least about 20 wt% water; (b) cooling and or evaporating the bottoms, and optionally adding an antisolvent to the bottoms, to attain a temperature and composition sufficient to cause the bottoms to separate into a Ϊ-Ι4 OOG-R-COO ^' ¾ ⁺ -containing liquid portion and a N¾ ^{T "} OOC~R-COOH-containirig solid portion that is substantially free of ¾ ⁺ OOC-R-COO ^' NH ; (e) separating the solid portion from the liquid portion; and (d) recovering the solid portion,

[0006] We also provide a process for making HOOC-R-COOH from a NH * O C-R- COO ^" ΝΕ -containing fermentation broth, including (a) distilling the broth to form a first overhead that includes water and ammonia, and a first liquid bottoms that includes N¾* ^' OOC-R-C OH, at least some NH ^" OOC-R-COO ^" NH ₄ ⁺ , and at least about 20 wt water: (b) cooling and/or evaporating the bottoms, and optionally adding an antisolvent to the bottoms, to attain a temperature and composition sufficient t cause the bottoms to separate. into a NH ₄ ⁺ OC-R-COO ^" N¾ ^÷ -containing liquid portion and a NH OOC-R-COOH- eontaimng solid portion ^' that is- substantially free of N¾ ⁺ OOC-R-COO ^" N¾ ⁺ ; (c) separating the solid portion from the liquid portion; (d) recovering the solid portion; (e) dissolving the solid portion in water to produce an aqueous NH* ^{+ "} OOC-R-COOH solution; (fj distilling the aqueous NH ₄ * OOC-R-COOH solution at a temperature and pressure sufficient to form a second overhead that includes water and ammonia, and a second bottoms that includes a major portion of HOOC-R-C OH, a minor portion of NH ^' OOC-R-COOH, and water; (g) cooling and/or evaporating the second bottoms to cause the second bottoms to separate into a second liquid portion in contact with a second solid portion that preferabl consists essentially of HO C-R-COOH and is substantially free of N¾ ^{+ "} OOC-R-COOH; (h) separating the second ^' solid portion from the .second liquid portion; and (i) recovering the second solid portion.

[0007] We further provide a process for making NH ₄ ^{+ "} O C-R-COOH from a clarified N¾ ^{÷ "} OOC-R-»COOH-containing broth including (a) optionally, adding NH ₄ ^{'+ "} OOC-R- COOH, ^" OOC-R-CO ^" l-lT, HOOC-R-COOH, NH ₃ , and/or NJ¾ ⁺ to the broth to preferably maintain the pH of the broth below 6; (b) distilling the broth to form an overhead that includes water and optionally ammonia, and a liquid bottoms that includes NH ₄ ⁺ OOC- R-COOH, at least some NH ⁺ OOC-R-C O ^" N¾ ⁺ _; and at least about 20 wt% water; (c) cooling -and/or evaporating the bottoms, and optionally adding an antiso!vent to the bottoms, to attain a temperature and composition sufficient to cause the bottoms to separate Into a NRf ^" OOC-R-COO ^* Nl -containing liquid portion and a NH ^" OOC-R-COOH-containtng solid -portion thai is substantially free of ¾ ⁺ OOC-R-COO ^" N¾ ^* (d) separating the solid portion from the liquid portion; and (e) recovering the solid portion,

[0008] We further yet provide a process for making H0OC-R-COOH from a clarified N¾ ⁺ GC-R-COOH-containmg. fermentation brot including (a) optionally, adding NET ^" OOC-R-CO H, ¾ ⁺ OOC-R-COO ^" N¾ ⁺ , HOOC-R-COOH, N¾ and/or fLf to the broth to preferabl maintain the pH of the broth below 6; (b) distilling the broth to form an overhead that includes water and optionally ammonia, and a liquid bottoms that includes NH»* ^" OOC-R-COOH, at least some Ni ^" OOC-R-COO ^" N!¾\ and at least about 20 wt¾ water: (c) cooling arid/or evaporating the bottoms, and optionally adding an antisolveni to the bottoms, to attain a temperature and composition sufficient to cause the bottoms to separate into a Nl¾ ^{+ "} OOC-R-COO ^' NH* ⁺ -containtng liquid portion and a N¾ ^{+ "} OOC-R-COOB- containing solid portion that i substantially free of ΝΗ, OOC-R-COO ^" N¾ ⁺ ; (d) separating the solid portion front the liquid portion; and (e) recovering the solid portion; (f) dissolving the solid portion in water to produce an aqueous NH4* OOC-R-COOH solution; (g) distilling the aqueous MB** ^" OOC-R-COOH solution at a temperature, and pressure sufficient to form second overhead that includes water and ammonia, and a second bottoms that includes a major portion of H OC-R-COOH, a minor portion of NH, ^{+ "} OOC-R-COOH, and water; (h) cooling and/or evaporating the second bottoms to cause the second bottoms to separate into a second liquid portion in contact with a second solid portion that preferably consists essentially of HOOC-R-COOH and is substantially free of NH ₄ ^{÷ "} OOC-R-COOH; (!) separating the second solid portion from the second liquid portion; and (j.) recovering the second solid portion.

[0009] We additionally provide similar processes involving salts of the acids, For example, in the case of succinates, the salts can include rnonosodiu . succinate (M aS) when sodium (Na) is used, monopotassium succinate (MKS) when potassium (K) is used, or magnesium succinate (MgS) when magnesium (Mg) is used. Brief Description of the Drawings

[0010] Fig. 1 is a block diagram of a process for the production of ΝΗ ^" OOC-R-COOH from a fermentation broth containing ΝΗ ^" OOC-R-COO ^" NH_f , (0011] Fig. 2 is a flow diagram showing selected aspects of OUT process.

Detailed Description:

[0 12] ft will be appreciated that at least a portion of the following description is intended to refer to representative examples of processes selected for illustration in the drawings and is not intended to define or limit the disclosure, other than n the appended claims.

[0013] Our processes may be appreciated by reference to Fig. 1 , which shows in block diagram form one representative example 10, of our methods.

[0014] HOOC-R-COOH compound acids, NH OOC-R-COOH compounds and NH/ ^" OOC-R-COO ^" NH compounds; wherein R may be selected from Ihe group consisting of Oh, CH-CH, C¾~CH£OH), (CH ₂ ) ₃ , CH ₂ -C=CH _¾ CH-CH-CH^CH, iC¾)¾ and ( H ₂ )io _» for example, are referred to herein. Exemplary, HOOC-R-COOH compound acids include but are not limited to maionic acid, fumarie acid, malic acid, giutarlc acid, citracontc acid,, itaconic acid, mueonfc acid,, sebaeic acid and dodecanedioic acid; wherein R is selected from the group consisting of CH _2j CHKTH, CH CH(OH), (CH ₂ ) ₃ , C(CH ₃ )=€H, CH ₂ -C ^« C¾, CH-CH-CH-CFL (CH ₂ ) _S and (CH ₂ .) _!Q . Exemplary, NH OOC-R-COOH compounds ^' include but. are not limited to monoammonium mabnate, monammoniunr fumarate, monoammonium nialate, mondammon m glutarate,. monoammonium eitraeonate, monoammonium itaconaie, monoammonium muconate, monoammonium sebacate and monoammonium dod ^' ecanedionate; wherein R Is selected from the group consisting of CH ₂ , CH ₂ -CH(OH), (CH ₂ ) ₃ , C<CH ₃ ) ^» CH, CH ₂ -C=CH ₂ , CH-CH-CH-CH, (CH ₂ ) _S and (C¾)ie. Exemplary, NHf OOC-R-COO ^" NH/ compounds include but are not limited to diammeiuum malonate, diammonium fumarate. diammonium ma!ate, -diammoniu glutarate, diammonium eitraeonate, diammonium itaconate, diammonium muconate. diammonium sebacate and diammonium dodecanedionate: wherein R is selected from the group consisting of CH≥, CB-CIi CHrCB(OH), (CH ₂ ) ₃ , C(e¾)=€B, CH ₂ -C<%, CH-CH-CH-CH, (C¾)s and iC¾) _!0 .

[0015] -A growth vessel 1 , typical ly an in-place steam steriii ^' zabie fermentor, may he used to grow a microbial culture (not shown) that is subsequently utilized for the production of the NH/ ^" OOC-R-COO ^* NH/ compound, NH/ ^' OOC-R-COOH compound, and/or HOOC-R- COOH compound acid-containing fermentation broth. Such growth vessels are known in the art and are not farther discussed. 00:16] The microbial culture may comprise microorganisms capable of producing H ₄ ^{+ "} OOC-R-COO ^" H ₄ ⁺ compounds/! IOOC-R-COOH compound acids from fermentable carbon sources such as carbohydrate sugars (e.g.. glucose), cyclohexanol, alkanes (e.g., n-aikanes), plant based oils arid others. Representative examples of microorganisms include Escherichia coil (£. coil), Aspergillus niger, Aspergillus ierreus, Aspergillus itaconicus, Corynebacteri m glutamicu {also called Breviiweterhmrfiavutn), Enterococcus faecalis, Veittonella parvula, ActinobacMus succittogenes _* Paecilomyms varioti, Saccharomyces cerevisiae, Candida troplcalis, Bacteroides fragilis, Bacteroides rumimcala, Bacteroides amylophilus, Klebsiella pneumoniae, Phanerochaeie chrysorportum, Corynehacierium nilri!opMltis, Gordona ierrae, Rhodococeus rhodochrous, Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Rhadotor la mitcilangmosa, P eudomonas putida, Candida mahosa, Rhizopus species (spp.) such as Rhizopus nigricans, Rhizopus arrhizus, Rhizopus oryzae, Rhizopus aligosporus, Rhizopus microsporous, Rhizopus eircinans, Rhisopiis formosa; M c r spp.; Cuninghamella spp.; Circinelh spp.; Lactobacillus, -spp. mixtures thereof and the like. Additionally, other microbes such as Escherichia spp., Aspergillus spp., Uslilago spp., Corymhacterium spp. (also called Brevibacteriumflavum), Enterococcus spp., Veillonella spp.. Acimabac lus spp., Paeci myces spp., S ^' accharomyces spp., Candida spp., Bacteroides spp., Klebsiella spp., Phanerochaeie spp., Gordona spp., Rhodococeus spp., Rhadotorula spp. and Pseudomonas spp, as well as Toruiopsis spp,, Debaryomyees spp., Ha emda spp. -and P c/wa spp. may be used, as appropriate, in the preparation of fermentation broths containing Nl¾ ⁺ OOG-R- €00 ^' NH compounds.

[0017] Preferred microorganisms for diam omum fumaraie furaaric: acid production include Rhizopus oryzae Went et Prinsen Geeriigs, teleomorph strain NRRL 1526 having ATCC accession number 10260 (also known as Rhizopus arrhisus NRRL 1526); Rhtzop oligospor Saito, te!emorph strain NRRL 2710 having ATCG accession number 22959 ^; Rhizopus microspores van Tieghem, te!emorph deposited at Rhizopus cohnii Beriese ei De Toni, teleomorph strain U-l having ATCC accession number 464 ; . Rhizopus circinans van Tieghem, teleomorph strain NRRL 1474 having ATCC accession number 52315; Rhizopus ■oryzae Went et Prinsen Geeriigs, teleomorph strain NRRL 2582 haying ATCC accession number 52918; Rhizopus oryzae Went et Prinsen Geeriigs, teleomorph strain NRRL 395 having ATCC accession number 9363; and Rhizopus oryzae. Went et Prinsen Geeriigs, teleomorph deposited as Rhizopus stolonifer (Ehrenberg : Fries) Lind, teleomorph strain designated Waksman 85 having ATCC accession number 13310. Other microorganisms suitable for fumarie acid production include Lactobacilli host strains lacking the maloctate enzyme, fumarase and fumarate dehydrogenase. Such microorganisms can produce produce fumarie acid from monosaccharides such as glucose, sucrose, fructose and xylose; disaccharides such as maltose-; polysaccharides Such as starches and other carbon sources such as molasses, invert hig test molasses, syrups, grains, malted grains, cereal products, starch hydrolysate, com steep liquor and the like, Additionally, these microorganisms can produce fumarie acid when cultured on solid mediums such as potato dextrose .agar (PDA; ATCC medium 336) or its fluid medium equivalents,

[0018] Fermentation broths containing, fumarie acid can be produced from the Rhizopus oryzae- Went et Prinsen Cseerligs, teleomorph strain N L 395 having. ATCC accession number 9363. or other such strains, by culture in a sterilized liquid medium comprising 100 g/L glucose, 0.6 g/L urea, 0.5 mS/L com steep liquor, 03 g L B ₂ FO ₄ , 0.4 g/L MgSO,«*71¾0; 0.044 g/L ZnS0 ₄ »7H ₂ 0, 0.01 g/L ferric tartrate, 100 g/L CaC0 ₃ ; 300 ug mL poiyoxyetbylene sorbitan monooleate. ( ^' TWEEN 40™) and 300 u¾ mL polyoxyethy ne sor itan monopalrmtate (TWEEN 20™) In water for 4 days at 32?C as described In US 4,564,594 the subject matter of which is incorporated herein by reference.

[001-9] Fermentation broths containing- fumarie acid can be produced from Rhizopu oryzae strains by culture in a sterilized liquid medium prepared by adding 15 g invert sugar produced by enzymatic or acid inversion of high test niola ses. 9.75 g calcium carbonate, 0.0375 g magnesium sulfate heptahydraie; 0.15 g potassium acid phosphate, 1 .5 mg ferric sulfate and about 0.02 % (w/w of the liquid fermentation culture medium) to about 0 ^, 25 % (w/w of the liquid fermentation culture medium) of either ammonium sulfate or urea in 140 m.L of water and sterilizing the medium. This sterilized liquid medium is then inoculated with Rhizopus oryzae and the fermentation may be conducted for 7 days at ^' 28*C to 32°C with the addition of about 0.02% (w/w of the liquid fermentation culture medium) to about 0.12% (w/w of the liquid fermentation: culture mediuro) of eithe ammonium sulfate or urea at about 24 h _t about 48 ^' h, about 72 h and about. 96 h after inoculation, or at combinations of these times, after inoculation as described in US 2,912,363 the subject matter of which is incorporated herein by reference.

[0020] Preferred microorganisms for d .ammonium maJonate/malonic acid production include Phanerochaete ehrysorporium BurdsalL teleomo ph strain V M F-1767 having ATCC accession number 24725™; Corynebacterium mtribphil s Akio et at. strain C42 having ATCC accession number 21419™ Gordona terme strain MA-I having National institute of Bioscience and Human-Technology (Higashi 1-chorae, Tsukuba-shi, ibarakiken, japan) accession number FE M-BP-453S; and R ^' hod co us rhodochro s (Zopf) Tsukamura emend, Rainey ei al. deposited as Noc rdia l cid strain i ^' MRU 3890 having ATCC accession number ^' 33025™ which may produce malonic acid, or malonic acid moooesters represented by the formula H00C-CH2-COOR' (where R' is an alkenyl, aryl araJkyl or C3-20 alky!), from monosaccharides such as glucose, polysaccharides such as starches and cellulose, and other carbon sources such as wood, corn and cyanoacetic acid esters represented by the formula NC-CHa-COOR' (where &' is an an alkenyl, aryi,. aralkyl or C3-20 alkyl). Additionally, these microorganisms may produce malonic acid when cultured on solid mediums such as potato dextrose agar (ATCC medium 336), yeast extract-glucose medium (ATCC medium 25), nutrient agar (ATCC medium 3) or fluid, medium equivalents of these or malonic acid esters when cultured in such mediums with cyanoacetic acid esters.

[0021] Fermentation broths containing diammonium malonate/malonic acid ma be produced from the Phamroch eie chrysorporium Burdsall, teteomorph strain VKM F-1767 having ATCC accession number 24725™. or other such strains, by culture in a liquid medium comprising a 1% carbon source of glucose, cellulose or wood as a liquid stationary culture at 37 C as described by Abbas et a 47 Curr. Genet. 49 (2005) the subject matter of which is incorporated herein by reference, that is flushed with water-saturated C¾ on day 2 after inoculation and every 3 days thereafter. The production of diammonium malonate/maionie acid by Ph nerochaete chrysoparium is also described in National Laboratory Field Work Proposal Final Report having FWP/OTIS Number: DE-AC06-76RL0 1830 #41721 and by Kragsley, MT; Rornine, RA; and Lasore, LL "Effects of Medium Composition on Morphology and Organic.; Acid Production in P ^' hanerach ete chrysosporium"- in a poster presentation at the Annual Meeting of the American Society of Microbiology the subject matter of which is incorporated herein by reference,

[0022] Fermentation broths containing malonic acid esters may be produced from G rd na terme strain MA-1 having National Institute of Bioscience and .Human-Technology (Higashi 1 -chome, Tsukuba-shi, Ibarakiken, Japan) accession number FERM-BF-4S3S, Corynebacterium- ^' nitrihp ilm Akio et al. strain €42 having ATCC accession number 21 19™ or Rhodococcus rhodochn s (Zopi) Tsukamura emend. Rainey et al deposited as Rocardia lucida strain IM U 3890 having ATCC accession number 33025™ by inoculation of one of these microorganisms into 3 ml of sterilized LB medium { 1% polypeptone, 0,5% yeast extract 0,5% Na ^' Cl) and culture at 30°G for 24 h with shaking. One mi of the resultant cell culture liquid may be inoculated into 100 ml of a sterilized medium A (pH 7.2) comprising glycerol (1.0%), isovaieroniirUe <0.2%} _} yeast extract (0.02%), ¾P0 ₄ (0,2%), MgS0 »7¾0 (0,02%), FeS0 ₄ ®7H ₂ 0 ( 10 pjpm), CoCI ₂ «4H ₂ 0 (10 ppm), CaCi ₂ ®2¾0 (I ppm) and n€!2®4l¾Q (7 ppnV) and cultured at 30°C, for 48 hrs. After completion of the cultivation, -the culture liquid is centrifuged. The total volume of the resultant cell pellet is then washed with deionked water and suspended in 100 mi of 50 mM phosphate buffer (pH 7.0). Ideally, the turbidity of th cell suspension, is ODwonre - 5.5 - 5.6. To this cell suspension 1 ,00 g of ethyl cyanoacetate is added as a substrate, when using the Gordona (errae strain or the Corynebacierium mlrilopMlus strain, and reacted at 30°C for 1 h to form a fermentation broth containing monoethyi malonate; When using the Rhodococcm rhodochrous strain, 1.00 g of n-pfopyl cyanoacetate is added as a substrate and reacted at 30°C tor 1 h to form a fermentation broth containing mono-n-propy! maJonate. Analysis by high performance liquid chromatography (HPLC;. column: TS gel ODS-120A (Tosoh Corp.), 4.6 mm LD.x25 cm; mobile phase: 5% acetc hrile, 95% water, 0,1% phosphoric acid; flow rate: 0,5 ro!/min; detection: UV 220 nm) may be performed to confirm conversion of the ethyl cyanoacetate to monoeihyl malonate or the conversion of n-propyl cyanoacetate to-mono-n-propyl malonate.

[0023] After completion of the reaction,, cells may be removed from the fermentation broth by centrifugation. 2N HCl may be added to the resultant solution to adjust the pH to 2,0. Thereafter,, monoethyi malonate, the reaction product, may be -extracted from the broth with ethyl acetate. Anhydrous sodium sulfate may be added to the resultant organic layer for dehydration _* and the solvent can be removed by distillation to obtain monoeihyl malonate was at a yield of about 89.9%, The production of such maionic acid esters havin the formula HOOC-CH -COOR' from cyanoacetic acid esters represented by the formula MC- CHrCOO ' by Gordona terras strain MA-i having National Institute of Bioscience and Human-Technology (Higashi 1-chome, Tsufcuba-shi, !harakiken, Japan) accession number FERM-B ^' P-4535 and Corynebacierium nim philus Akio el at. strain C42 having ATCC accession number 21419™ is also described in ^" 1 ⁾ 86,238,896 the subject matter of which is incorporated herein by reference.

S [0024] Alternatively, after completion of the reaction at 30°C for ! h and the removal of cells, a saponification reaction may he performed by treating, the fermentation broth from the Gordana ierrae strain or the Coryneb cMri m nUHlophilvs strains with a quantity of a strong base such as KOH sufficient to remove the monoethyi group from the monoethyl malonafce ester and to form the alcohol CM ₃ -C¾~OH (ethane!) and HOOC-CH ₂ -COO\ The resulting HOOC-C¾-COO ^" may then be treated to form the appropriate fermentation broth

[0025] Similarly, after completion of the reaction at 30°C for _. I h and the removal of cells, a -saponification reaction may he performed by treating the fermentation broth from the Rhodococeus rhodochrous strain with a quantity of a strong base such as KOH sufficient to remove, the mono-n~propy1 group from the mono-n-propyl ma!oaate ester and to form the alcohol CH3-CH ₂ -CH ₂ -OH (n-propanol) and LiQOC-CH COO ^' (which may be in the form of a salt). The resulting HOOC-CH2-COO ^" may then be treated to form the appropriate fermentation broth.

[0026] This approach of producin fiOOC-CH ₂ -C0OR' esters In fermentation broths followed by de-esterification to form HOOC-CHa-COO ^" and R ⁵ -OH via saponification, reactions, and the like to produce a -fermentation broth can al o be used with malonate esters produced i ons other cyanoacetic acid esters represented by the formula NC-CH2-COOR'.

[0027] Preferred microorganisms for diammo um raalonate/malic acid production include Aspergillus flavtis Link, anamorph strain A- 1 .14 having ATCC accession number 1 _. 3697™ ^· ; Aspergillus fiavus Link, anamorph strain A-57 having ATCC accession number 13698™; Aspergillus parasiticus Speare, anamorph strai WB 465 having ATCC accession number 1 869™; Aspergillus parasiticus Speare, anamorph strain A-237 having ATCC accession number 13696™; and Aspergillus oryz (Ahlbnrg) Cohn, anamorph strain NRRL 3488 having ATCC accession number 56747™ which produce dlammomurn malonate malic acid from monosaccharides such as glucose, sucrose, fructose, galactose sorbose and xylose; disaccharides such as maltose; polysaccharides such as starches and other carbon sources such as sorbitol, glycerol, molasses, corn and the like. Additionally, these microorganisms may produce diammonlum malonate/malie acid when cultured on solid mediums such as Czapek's agar (ATCC medium 3 12} and potato dextrose agar (PDA; ATCC medium 336) or fluid medium equivalents of these.

[0028] Fermentation broths containing diainmonium mafonate maiic acid may be produced from the Aspergillus fiavus Link, anamorph strain A-l 1-4 having ATCC accession tiumber 13697™, Aspergillus flavus Link, anamorph strain A-57 having, ATCC -accession number 13698™, Aspergillus parasiticus Speare, anamorph strain WB 465 having ATCC accession number 16,869™, Aspergillus p - arasiticus Speare, anamorph strain A-2.37 having ATCC accession number 13696™ ^' and. Aspergillus oryzae (Ahlburg) Cohn, anamorph strain NRRL 3488 havin ATCC accession number 56747™, or other such strains, by aerobic culture, in I L of a liquid medium comprising 10% glucose, 0,6% peptone, 0,015% Κ¾Ρ0 , 0.015% K ₂ HPC¾, 0,01% MgS0 ₄ »7H ₂ 0, 0,01% -CaC_- ₂ *2%0, 5 mg of aCI, 5 mg of FeS04*7.H20 and distilled water (all percentages here are by weight per volume, i.e. grams per cubic centimeter).. The 1 L solution is then divided into 30 ml, portions each of which Is placed into a separate 250 m flask and is sterilized by heating, under pressure at !20°C for 15 min. 4% of CaC<¾ (the percentage here is by weight per volume, I.e. grams per cubic centimeter), which was separately sterilized by dry heating, is then added to each of the flasks. Thereafter a microorganism such as. Aspergillus flams Link, anamorph strain A-11 having ATCC accession number 13697™.. Aspergillus flav Link, : anamorph strain A-57 having ATCC accession number !369-8™ _¥ Aspergillus parasiticus Speare, anamorph strain WB 465 having ATCC accession number 16869™. Aspergillus parasiticus Speare, anamorph strain A-237 having- ATCC accession number 13696™ and Aspergillus oryzae (Ahlburg) Cohn, - anamorph strain NRRL 3488 having ATCC accession number 56747™, or other such strains or combinations of these, may be Inoculated into- the culture solution. The microorganisms are then cultivation for 7 days at 28°C on a rotary shaker operating at 200 rpm to produce a fermentation broth with a pH of about 5.4 to about 6.2 that, contains malic acid (L-malic acid). Typically, the fermentation broths produced- may contain from about 21 ,8 mg/mL to about 32.6 mg mL of L-malic acid. The production of fermentatio broths from Aspergillus flavus, Aspergillus parasiticus and Aspergillu oryzae in the medium- described above, and other mediums such as solid equivalents of these, is also described in US 3 ,063.91 the subject matter of which is incorporated herein by reference,

[0029] Preferred microorganisms for diamraonium gHitarate/giutarie acid production include Candida iropicalis (Casteiiani) Berkhout, anamorph strain OH23 having, ATCC accession number 24887™, Rhodotonda mucilanginosa (Jorgensen) Harrison var. mucilaginasa, anamorph deposited as ^* Rhodotonda rubra (Demme) Lodder, anamorph strain AKU 4817 having ATCC accession number 64041™ and the lysine-requir ^' ing Saccharomyces cemvisiae strain C-l which produce glutarie acid from monosaccharides such as glucose and other carbon sources such as azelaic acid, n-psntadecane, and the like. Additionally, these microorganisms can be cultured on mediums such as malt extract agar (Blakesiee's formula; ATCC medium 325); YM agar (ATCC medium 200) or YM broth (ATCC medium 200) or equivalents of these,

[0030] Fermentation broths containing diammonium glaiame/glutar!c acid may be produced from the Candida tropicalis (Casiellani) Berfchout, anamorph strain OH23 having ATCC accession, number 24837™, or other such strains, by culture at 32°C in a liquid medium containing 300 mg of NH ₄ B. ₂ PG ₄ , 200 mg ofKH ₂ P ⁽ ¼, 100 mg of K ₂ HP0 , 50 mg of MgS0 ₄ *7H ₂ 0, 1 μ$ of bi tm, 0.1% (w/v) yeast extract and about i % (wV) »-pentadecane in 100 mi of distilled water. The procedure for producing fermentation broths from media containing Λ-pentadeeane by catering Candid tropica! is (Casiellani) Berfchout anamorph strain OH23 having ATCC accession number 24887 is also described in Okuhura et a!., 35 Agr. Biol Chem, 1376 (1.971.) the subject matter of which is .incorporated herein by reference.

[0031 ] Fermentation broths containing diamrnonium gSutarate/glutarie acid can be produced from the Rhod&torulu mucilang as (Jorgensen) Harrison var. mucit ginosa, anamorph deposited as Rh dotor t rubra (De me) Lodder, anamorph strain AKU 4817 having ATCC accession number 64041™, or other such strains, as follows. The Rhadotonda mucila gm sa strain is cultured at 28°C for 38 h with shaking in a 500 mi flask containing 100 mL of a liquid medium comprising 1.0% glucose, 0.5% peptone, 0.3% malt extract and 0.3% yeast extract (all percentages here are by weight per volume, e. grams per cubic centimeter). The initial pH is then adjusted to 6.0. After growth, cells are harvested and washed three times with saline solution and then suspended in M 200 potassium phosphate buffer (pH 7.5). The cell suspension (about 20-40 mg/nif) is then incubated with aze!aie acid (4 mg/ ^' mL) in 3 ml of 0.2 M Tris-HCI buffer (pH 7.5) at 28°C for 24 h with shaking. Ceils are then removed from this fermentation broth by centrifugation to produce a clarified fermentation broth. The production of fermentation broths from Rhodotoruta mudtangmosa strains according to the process described above is also described by Ohsugi et at,.48 Agric. Biol Chem. 1881 ( \ 984) the subject matter of which is incorporated herein by reference.

[0032] Fermentation broths containing diammonium glntarate gHitaric acid may be produced from the iysine-requinng Sacch romyces cerevisiae strain C-l as follows. A sterilized medium- having a pH of 5.5 and comprising 75 g glucose,. 7.5 g NB H2PQ4, 3 g XH2PO4, 0.183 mg MgS0 ₄ ^« 7H ₂ 0, 1.13 mg nicotinamide, 0.15 g biotin, 3.8 mg calcium pantothenate, 75 mg i -inositol, 33 mg thiamine hydrochloride, mg pyridoxine hydrochloride, 13,2 mg £nS0 ₄ ^» 7H ₂ 0, 7.88 mg FeSO NK aSC eHsO, 0.73 mg GuS04 ^« 5H ₂ 0 and 100 mg L-iys;ne*HC! in 1 L of water is prepared, The medium is then distributed in 200 ml portions into 500 ml-Erlenmeyer flasks which are inoculated with the ! sine-requiring Saccharomyces cerevisiae strain C-L The lysine-requiring Saccharomyces cerevisiae strain C-l is described by Mattooh el al, 51 Biocbim. Et Biophys. Acta 615 (19 1),. the subject matter of which is incorporated herein by reference and was derived from the Saccharomyces cerevisiae C strain. Fermentations may then be performed at 30°C on a rotary shaker for 9 days after which cells are removed from the fermentation broth by cenirifugation. The resulting fermentation broth- may be reduced 10 fold under reduced pressure at 35°G. The production of fermentation broths from the !ysihe-requiring Saccharomyces cerevisiae strain C-l. according- to the process described above is also described by Matron and Haight 237 J. B io!., Chem. 3486 (1962) the subject matter of which is incorporated herein by reference.

f 033] Preferred microorganisms for diammonium itaconate itaconic acid production include Aspergillus terreus Thorn, anamorph strain NRRL 3960 having ATCC accession number 10020™; Aspergillus terreus ^' strain RC4' derived from the NRRL I960 strain; Aspergillus terreus strain CM 85 J derived from the NRRL 1960 strain; Aspergillus terreus Thorn, anamorph strain NRRL 265 having ATCC accession number 10029™; Aspergillus- terreus "Thorn, anamorph strain. F4845 having ATCC accession number 20542™; Aspergillus terreus Thorn, anamorph strain K 26 having ATCC accession number 32359™; Aspergillus terreus Thorn, anamorph strain 14/11 having ATCC accession number 32587™; Aspergillus terreus Thorn, anamorph ^' strain 21/1 having ATCC accession number 32588™; Aspergillm terreus Thorn, anamorph strain 25/ί ^' Π having ATCC accession number 32589™; Aspergillus terreus Thorn, anamorph ^' strain K having ATCC accession number 32590™; Aspergillus terreus Thorn, anamorph strain 3 having ATCC accession number 36364™:. Aspergillm terreiis TN484- .1 ; Aspergillus itaeomc Kinshita, anamorph strain NRRL 363 having ATCC accession number 56806™; and Basidiomyeetes of the genus Usiilago which produce diammonium itaconate/itaconic acid from monosaccharides such as glucose, sucrose, fructose, xylose and arabinose: d.isaccharides such as lactose; polysaccharides such as starches and other carbon sources such as molasses, sago starch, sago starch hydroiysate, corn steep liquor and the like. Additionally, these microorganisms may produce diammonium itaconate/liaconic acid when cultured on solid mediums such as Czapek*s agar (ATCC medium 3 2), potato dextrose agar (PDA; ATCC medium 336), malt agar medium (ATCC medium 323), malt extract agar .(Biakeslee's formula; ATCC medium 325); malt extract agar (ATCC medium 324) potato dextrose yeast agar (PDY; ATCC medium 337) or fluid medium equivalents of these.

[0034] Fermentation broths containing diammonium itaconate/itaconic acid may be produced from the Aspergillus terreus strain TN484 strain, or other such strains, by culture in a liquid medium comprising 140 g/L sago starch hydro ^' lysate, L-8.g L corn steep liquor, 1.2 g/L MgS0 »7H;0 and 2.9 g/L NFLJNGJ in water as described by Dwiarti et l. 98 Bioresour. Technoi . 3329 (2007) the subject matter of which is incorporated herein by reference,

[0035] Fermentation broths containing diammonium itaconate/itaconic acid may be produced from the Aspergillus iermm strain RC4' derived from the NRRL- 1960 strain and Aspergillus terre s strain CM85J derived from the NRRL i960 strain by culture at 35°C in a liquid medium comprising glucose as described in French patent 8805487 and by Giiiet et al., 1771 Bact. 3573 (1 95) the subject matter of which is incorporated herein by reference.

[0036] Fermentation broths containing diammonium itaconate/itaconic acid may be produced from the Aspergillus terreus Thorn, anamorph strain MF4845 having ATCC accession number 20542™ by culture in a liquid, lactose based medium (LBM) or an identical medium containing, glucose instead of lactose as described by Lai et al.,. 104 I. Biosci. Bioeng. 9 (2007) the subject matter of which is incorporated herein by reference, [0037] Fermentation broths containing diammonium itaconate/itaconic acid may be produced from the Aspergillus terreus Thorn, anamorph strain NRRL 1 60 havin ATCC accession number 10020™ by culture in an aqueous medium. This -aqueous medium may be prepared by making an aqueous solution of wheat starch having 350 g L of dry solids by homogenization, adjusting the pH to 6.5 and adding 0.175 g/L of the -liquefying -.enzyme (TERMAMYL 120L® marketed by Novozyroes A/S, Denmark), This aqueous solution may then be introduced into a steam injection sterilizer. The temperature may then- be maintained at l Q0 ^a C to lOS^C for 7 mm, cooled to 95°C and may be maintained at this temperature for 2 hours in an agitated tank and then, cooled to 35°C to produce a fluidszed starch solution. Next, a quantity of the fiuidized starch solution- containing 130 kg of dry solids may be introduced into a 1 ,400 L fermenter. The following materials may then be added to this fermenter:

(i) a nutritive solution sterilized for 30 mm at 100 C, containing 0,5 kg com extract, 3,45 kg magnesium chloride, 0.3 kg magnesium- sulfate, 0.9 kg urea, 0.4 kg sodium chloride. 0,033 kg zinc sulfate, 0.05 kg raonopotassiurn phosphate, 1 kg calcium chloride, 0,06 kg copper sulfate and 0.3 kg sulfuric acid (the pFI was 3.6).; and

(is) 0.29 kg amylogiucosidase (AMG 200 L , marketed by Novo Industry- marketed by Novozymes A S, Denmark),

[0038] This production medium, the final volume of which may be adjusted to 1 ,000 L with sterile water, may then be agitated, aerated and inoculated with 20 L of Aspergillus terreus Thorn, anamorph strain NRRL 1960 having- ATCC accession number 10020™ culture,, aged 35 hours- that may be previously prepared at 32 to 35 ^C' C in a fermenter containing 25 g/L glucose, 4.5 g/L magnesium sulfate, 0.4 g/L sodium chloride, 0.004 g L zinc sulfate, 0.L g/L monopotassium phosphate, 0,5 g/L corn extract. 2.0 ammonium nitrate and 0,5 g/L sulfuric acid. The temperature of the medium in the inoculated 1 ,400 L fermenter may then be -maintained at 32 ^W C to 35°C and the fermentation may be terminated after the sugar was consumed and when ^' the acidity is maximal and stable. Wort samples maybe periodically withdrawn from the 1,400 L fermenter to evaluate the itaconic acid content. This method of d ^' iammonium itaconate/itaconi-c acid production from Aspergillus terreus Thorn, anamorph strain NRRL 1960 having ATCC accession number 10020™ culture and similar methods for diammomurn itaeonaie/itaconic acid production are also described in US 5,231,016 the subject matter of which is incorporated herein by reference.

[0039] fermentation broths containing diamrooamm Itaconale/itacomc acid may be produced from the Aspergillus terreus Thorn, anamorph strain NRRL 1 60 having ATCC accession number 10020™ by culture at 3 °C to 35°C in an aqueous medium comprising between Ό g to 1 10 g of sucrose and 0 g to 100 g of glycerol, 0,5 g of maize extract (CSL; corn steep liquor), 1.2 g of ammo ium nitrate, 0.3 g of hydrated magnesium sulfate, 0.3 g of magnesium oxide, 0.315 g of calcium hydroxide, 0.05 g of monopotassium phosphate and 0.380 g of hydrated copper nitrate in 1 L of water and the pH of the medium may be adjusted to a value on the order of 2.8 to 3 with a nitric acid solution. ^' The fermentation ma be terminated after the sugar has been exhausted and when the acidity of the medium is at a maximum and stable. Samples of -musts may be- taken near the end of the fermentation to evaluate the fermentation broth. This method of diammo um ifaconate/iiaconic acid production from Aspergillus (emus Thorn, ana orph strain NR L i 960 having ATCC accession number 10020™ culture and similar methods- for itaconic acid production are -also described in US 5457040, US 3873425 and by agnuson and Lasure eds., Advances in Fungal Biotechnology for industry-, Agriculture, and Medicine. Chapter 12: Organic Ad Production in Filamentous Fungi, pages 307-340; Kluwer Academic/Plenum Publishers (2004). the subj ect matter of which is incorporated herein by reference,

[0040] Fermentation broths containing diarnmonium citraconate citraeonic acid can be produced from fermentation broths containing i laconic acid as follows. A first fermentation broth containing about 5% (w/w) to about 20% (w/w) itaconic acid may be prepared as described above, !f necessary the first fermentation broth may be concentrated or diluted to obtain these, or other desired, itaconic acid concentrations. Cells may be removed from the first fermentation broth by eentrifugation, filtration or other means. The- itaconic acid may then fee placed in free acid form, if necessary, by treatment of the first fermentation broth with, an acid. Typically, pFI may be between 1.5 and 5 and most preferably may be between 2 and 2.5. The first fermentation. brot& may then Optionally be concentrated at about 50°C to about 100°C or at a pressure of about .100 mm to about 500 mm Hg.. Preferably, the first fermentation, broth obtained has an itaconic acid concentration of from about 20% (w/w) to about 80% (w/w), or more preferably from about 30% (w/w) to about 50% (w/w), relative to the weight of the broth. A solvent that is sparingly soluble in water and. which forms an azeotrope when added, such as pseudocumene or c clohexanone, and a catalyst, such as pyridimu phosphate, may then be added to the first fermentation broth to dehydrate and isomcrize the itaconic acid to citraconic anhydride. The resulting second fermentation broth comprising citraconic anhydride, solvent and possibly other fermentation broth components may then be contacted with an aqueous solution at an appropriate pH so that the citraconic anhydride may be hydro!yzed to citraconic .acid. In this manner, a third fermentation broth may be produced for use in the production of citraconic acid.

[0041 ] For example, 2000 kg of an Aspergillus spp, mycelium-free, first fermentation broth having an itaconic acid concentration of 9.1 % (w/w) relative to the weight of the broth may be obtained. Concentration to 108"C may be performed to distil! off, and remove, essentially all of the water from the first fermentation broth. An equal weight of pseudocumene solvent and 5 g of pyridinium phosphate catalyst may then be added to the first fermentation broth. Any remaining water may be distilled off .at atmospheric pressure until no more eomes oft The resulting second fementatfon broth may be an organic solution containing 28.2 g of cirraconic anhydride with a yield based on the itaconic acid content of the first fermentation broth of 1 8%. Alternatively, 3% (w/ ) concentrated sulphuric acid relative to the weight of the itaconic acid in the first, fermentation broth may be added to the first fermentation broth prior to concentration and the other steps described above ar identically performed ^' to produce an organic solution containing .1 19 g of citraconic anhydride with a yield of based on the itaconic acid content of the first fermentation broth of 76%. The production of second fermentation broths containing citraconi anhydride from a first fermentation broth produced by Aspergillus spp, culture, or the culture of another itaconic acid producing microorganism, is also described in US 5,824,820 the subject matter of which is incorporated herein by reference. Such second fermentation broths comprising citraconic anhydride, solvent and possibly other fermentation broth components may then be contacted with an aqueous solutio at an appropriate pH so that the citraconic anhydride ma be hydrolyzed to citraconic acid. In this manner, a. third fermentation broth may be used for the production of citraconic acid.

[0042] Fermentation broths containing diammonium mueonate/myconic acid may be produced from the Pseudomon s putida (Trevisan.) Miguia strain MW 121 1.12 having ATCC accession number 3 V91 ^' 6™, or other such strains, by aerobic culture as follows, inoculums of Pseudomonas putida (Trevisan) Miguia strain MW 121 1.12 having ATCC accession number 31916™ are prepared in 250 ml shake flasks containing 50 ml of a sterilized liquid medium designated NO comprising 20 mM of sodium succinate, _. 7.1 g/L of NajHPO*. 3.6 g L H ₂ P0 _4i 2.25 g/L (NH ) ₂ S.04 _S 0.246 g/L MgS0 *7H _a Q, 0.0147 g/L of CaCb®2B _a O, 0.00278 g/L of FeS0 *7HsO and distilled water. The inoculum ma be prepared b culture at 30°C with shaking at 250 rp ^' fn for about 20 to about 24 hours to a turbidity of 200-240 kfeft units. A fed-batch fermentation may be conducted in a 16 L fennentor containing 12 L of a sterilized, liquid medium designated LP-! having an NaOB adjusted pH of 6.9 and comprising 20 mM of sodium acetate, 0,426 g/L of NajHP0 ₄ , 0.817 g/L K.H ₂ PC , 1.12 g L (NH ₄ ) ₂ S0 _4! 0,738 g L gS0 ₄ *7H ₂ , 0,0294 g/L of Ca<¾«2B ₂ 0, 0.0167 g/L of FeS0 ₄ ®7I-LCi _> 3.9 g/L acetic acid and distilled water. The fermenier may be inoculated with 150 mL of inoculum and toluene may be. supplied to the fermentation medium in vapor phase via air stripping at an air toluene vapor flow rate of 125 ee/min. The fermentation temperature may be maintained at 30 ^l 'C. pH may be maintained at 6,9 with 10 M Ν¾ΟΗ ^' and I M H ₂ S<¼ solutions, dissolved oxygen -may be maintained at 30-90% saturation with 600 PM agitation and 5 iiter/min aeratio (or approximately 0.5 VVM). PLURONlC ^'!'i¾ L61 pofyol (BASF) may be used as an antifoam agent. As the turbidity of the fermentation medium reaches 90-1 10 .kletf units (about 9-15 hours after inoculation}, an aqueous solution containing 10% (w/w> acetic acid, 0.1 14% (w/ ^' w) Na≤HP(¾ and 0.218% (w/w) KH2PO4 may be added to the fermen ^' tor medium at a rate of 0.4 mL min. Hie air-toluene vapor rate may then be increased to 250 cc/rniti and increased again to 500 cc min as the broth turbidity reaches 250 klett units. The. air-toluene vapor rate may be eventually increased to 750 cc/min as the turbidity reaches 450-550 klett units and a muconic acid product concentration of 15 g l is achieved in the fermentation broth. The fed-batch fermentation is normally continued for 24-36 h. The resulting fermentation broth may then be filtered to remove cells with a RO ICON® hollow tube "cross-flow'* ultrafllter whic has a poiysutfone type ultrafiltration membrane (P ^' M-100, molecular weight cutoff 100,000). The pH of the resulting clear, essentially cell-free fermentation broth may then, be adjusted to pH 1 -1.5 or another pH as desired, or necessary, with concentrated H2SO4.

[0043] A maximum specific productivity of 1.4 g gdw hr may be achieved during the fed- batch fermentation. The fermentation may be conducted by restricting the cell growth throughout the fed-batch fermentation cycle. During the early phase of fermentation (he,, 6- 12 hours ^' after inoculation of ceils),, the growth carbon source (20 mM or 1.2 g L acetate) and total phosphate (3 mM) may be in excess to initiate the growth of cells from 0.5-1.0 kiett unit (after inoculation) to 50-100 kiett units with 1-2 mM muconic acid concentration with utilization of the growth carbon source .and the phosphate. At this point, growth carbon source as well as the required phosphate level are fed to the fermentor to provide additional growth and enzyme induction. The production of fermentation broths containing muconic acid from Pse do on s putida (Trevisan) Migula strain Mf 121 1 ,12 having ATCC accession number 31916™, or other such strains in the medium described above and other similar mediums, is also described in US 4,535.059 the ^' subject matter of which is incorporated herein by reference.

[0044] Fermentation broths containing diammonium muconate/rauconic acid ma be produced from the Candida maiima Komagata et al, anamorph,. deposited as Candida cloacae Komagata ei al. anamorph strain AJ 4719 having ATCC accession number 20184™, or other such strains, by aerobic culture- as follows. The Candida ma!tosa Komagata et ai, anamorph, deposited as Candida cloacae Komagata et: a I., anamorph strain AJ 4719 having ATCC accession number 20184™ may be inoculated into a sterilized liquid medium comprising 1 g/L of catechol, 4 g/L of .NH NO¾ 4 g/L NaCi, 1,15 g L Na ₂ HP04, 0.2 g/L of ¾P0 , 0.1 g/L of C1, 10 mg/L of MgS0 ₄ «7M ₂ 0, 10 mg/L CaC! ₂ *2¾0), 5 mg L of FeS04®7¾0 and 500 mg L- of yeast extract in deJonized water. Cultures may then be grown at 30°C for several days and the fermentation broth may be centrifuged- to remove cells. The pH of the resulting essentially, cell-free fermentation broth may then be adjusted to about pH 2.0 or .another pH as desired, or necessary, with 2 N HCI. concentrated H ₂ S0 . The production of fermentation broths from the Candida makosa Komagata et ah, anamorph, deposited as Candida cloacae Komagata et al. anamorph strain AJ 4719 having ATCC accession number 20184™, or other- such strains, In the medium: described above is also described in Gomi and Horiguchi, 52 Agfic. Biol. Chum, 585 (1 88) the subject matter of which is Incorporated herein by reference.

[0045} Fermentation broths containing dian monium sebacate/sebacic acid may be produced as follows. Yeasts such as Candida spp,., Torulopsis spp,, Debaryctmyces spp., Hansenula spp. and Pichia spp. ma be cultured in sterile, mediums containing n-decane as the primary carbon source, such as a. modified YM broth (ATCC medium 200} lacking dextrose or equivalents ^' of ther yeast culture mediums known in the art containing n-decane as the primary carbon source, under standard culture conditions to produce a fermentation broth, it is preferred that _. fermentations ^' broths prepared by yeast culture be prepared from the culture of Candida tropicalis strains, such as Candida tropicalis ^' (Caste! lahi) Berkhout, anamorph. strain OH23 having ATCC accession number 24887™, in modified YM broth (ATCC medium 200} medium containing n-decane as the primary ^' carbon source and lacking dextrose. The production of fermentation broths from Candida tropicalis, or other such strain ^' s- and yeast species, is also described- in Ulezlo and Rogozhin, 40 Prikl Biokhim Mikrobiol. 533 (2004) the subject matter of which is incorporated herein by reference,

[0046J Fermentation broths containing diaramonium sebacate/sebacic acid may be produced as follows. An inoculum containing one !oopful of the yeast Torulopsis Candida strain NC-3-58 derived from Torulopsis Candid strain 99 grown on an slant of YM agar medium may be inoculated into 5 ml of a sterile, liquid -medium designated, decane medium that comprises 50 -ml n-decane, 2 g NFLH2PO ₄ , 4 g K2HPO4, 0.5 g MgSO-^l-LO, 1. g BACTO™ yeast extract, I rag η8θ4··ηΗ ₂ 0, ! mg.FeS0 ₄ »7H ₂ G and 1 mg ZnS0 ₄ «7¾0 in

950 ml of distilled water. Torulop. ccmdida strain NC-3-58 has a reduced bio-assimilation of sebacic acid relative to the Torubpste Candida strain 99 parent. The inoculum may then be cultured at 28°C for 2 days on a reciprocal shaker at 200 rpm with a stroke amplitude of 2 cm. Then 5 ml of the cultured broth may be inoculated into 66.5 mi of decane medium .in a 500 ml flask and cultured on. the same shaker at 28°C for 5-8 days. During the course of the fermentation, the pH of the medium may be kept at 7.5 by the addition of 2 N NaOH twice a day. A total of 1.5 L of inoculum broth should be prepared in this fashion. A sterilized, liquid medium having a pH of 7.0 and comprising 50 ml n-decane, 3 g Ν¾Η ₂ Κ> , 4 g K2HPO4, 0.2 g MgSt>4*nH_iO, 1 g BACTO™ yeast extract, i g; casamino acid, 1 mg MnS0 »nH ₂ 0, I mg ZnS0 ₄ *7H ₂ O, 1 mg FeSG4*7H ₂ 0 and 10 biotin in 950 ml of distilled water may then be prepared. This medium may also be modified to contain com steep liquor, malt extract, POLYPEPTON™, casamino acids, vitamin mixtures and combinations thereof. A 30 L jar _¾rn_enter (Marobishi MS3S-30L) containing 13,5 L of this medium may then be inoculated with 1.5 L of the inoculum broth. Culture may then be performed at an agitation rate of 600 rpm, a temperature of 25 ^e C and ^' a pH maintained at 6,5 for 83 h or longer (e.g., 6 days etc.) until the fermentation broth contains ⁽ he desired concentration. Fermentation broths may thus be obtained using this process. The production of fermentation broths from Torutopsis andida strain. NC-3-58 derived from Totulopsis Candida strain 99, and other such strains, is also described aneyuki et al., 58 J. Ferment. Technol. 405 { 1.980) the subject matter of which is incorporated herein by reference.

[0047] Fermentation broths containing diam omura sebacste/sebacie acid may be produced from the Candida tropic lis ^' strain having FER -P number 3291 , or other such strains, by aerobic culture as follows. The FERM-P number is an accession number assigned by the Fermentation Research Institute, Agency of industrial Science and Technology, at No, 5-2, 4-chome. Inagehigashi, Chiba-shi, Japan, from which the microorganisms having the FERM-P numbers are available. The tropkalis strain having. FERM-P number 32 i and the ability to produce a long-chain, dicarboxyiic acid from a straight-chain hydrocarbon may be cultured .in an. incubator containing an appropriate liquid medium, such as YM broth, to obtain 1 0 L of inoculum containing 10-15 g/L of cultured fungal body, and the inoculum ma be placed in. a reactor supplied with 1200 L of an. appropriate -sterilized culture- medium, such as a modified YM broth containing 240 L of n-decane (and optionally lacking dextrose). This medium may be mixed and adjusted -to pB 5 and cultured at 32°C for 12 h ₍ during which time sterile air may be supplied at a rate of 400 L min. Since the pH of the medium tends to drop during culture, a 10 N KOFI solution may be added as necessary to maintain the pi! of the medium at 5.0+/- 0.1. After 12 h, the pH of the medium may be shifted to 7.0 and the culture may be further continued for an additional 72 h. After this a fermentation broth of 1200 L containing diammonium sebaeate/sebaclc acid and cultured fungal body may be obtained. A bleaching powder and/or hypochlorite may then be added to about 50 ppm to the fermentation broth, stirred, exposed to the atmosphere and left under this condition for 5 days to avoid after-fermentation as desired, or necessary. The pH of the fermentation broth can similarly be adjusted as desired, or necessary. The production of fermentation broths containing dicarboxylte acid from straight-chain, saturated hydrocarbons, such as n-decane, as a substrate with the Candida tropkalis strain having FERM-P number 3291, or other such strains, is also described in US 4,339,536 the subject matter of which is incorporated herein b reference.

[0048] Fermentation broths containing diaramonium dodeeanediOnate/dodecanediotc acid may be produced from the Candida iropicaik strain having FERM-P number 3293 , or other such strains, by aerobic culture ^' as follows. The tropicatis strain having FERM-P number 3291 and the ability to produce a _. long-chairs dicarboxy!ie acid from a straight-chain hydrocarbon may be cultured in an incubator containing an appropriate liquid medium, such as YM broth, to obtain 1 0 L of inoculum containing 10-15 g/L of Cultured fungal body, and the inoculum placed in a reactor supplied with 1200 L of an appropriate sterilized culture medium, such as a modified YM broth containing 240 L of n-dodecane (and optionally- lacking dextrose). This medium may be mixed and adjusted to pH 5 and cultured at 32°C for 12 h, during which time sterile air may be supplied at a rate of 400 L/min. Since the pH of the medium tends to drop during culture, a 10 N KOH solution may be added as necessary to maintain the pH of the medium at 5.0+/- 0.1. After 12 h, the pH of the medium ma be shifted to 7.0 and the culture may be further continued for an additional 72 h. After this- a fermentation broth of 1200 L and a pH of 7.25 containing 42 kg/nr of dodecanedioic acid (IJO-decameihylenedlcarboxyilc acid), 0.9 kg/nr' of dodecano ^' ie acid, 1 1 kg m of h- dOdecanoie acid and 22 kg/m ³ of cultured fungal body may be obtained. A bleaching powder and/or hypochlorite may then be added to 50 ppm to the fermentation broth, stirred, exposed to the atmosphere -and left under this condition for 5 days to avoid after-fermentation as desired, or necessary. The pi I may similarly be adjusted as desired, or necessary. The production of fermentation broths from the Candida tropiealis strain having FERM-P number 3291, or other such strains, is also described in US 4,339,536 the subject matter of which is incorporated herein by reference.

[0049] A fermentable carbon source (e.g., carbohydrates and sugars), optionally a source of -nitrogen end complex nutrients (e.g., corn steep liquor), additional media components such as vitamins, salts and other materials that can improve cellular growth and/or product formation, and water ma be fed to the growth vessel 12 for growth and sustenance of the microbial culture. Typically, the microbial culture is grown, under aerobic conditions provided, by sparging an oxygen-rich gas (e.g., air or the like), Typically, an acid (e.g., sulphuric acid or the like) and ammonium hydroxide are provided for pH control during the growth of the microbial culture.

[0050] n one example (not shown), the aerobic conditions in growth vessel 12 (provided by sparging an oxygen-rich gas) may be switched to anaerobic conditions by changing the oxygen-rich gas to an oxygen-deficient gas (e.g., CO? or the like). The anaerobic environment may trigger biocon versio of the fermeMabie carbon source to a HO C-R- COOH compound acid in situ in growth vessel 12. Ammonium hydroxide is provided for pH control during bioeon version of the fermentable carbon source to the HOOC-R-CGOH compound acid. The H OC-R-COOH compound acid thai is produced is a least partially neutralized to the NH * OOC-R-COO ^" Nl¾ ⁺ compound due to the presence of the ammonium hydroxide, leading to the production of a broth comprising the H ₄ ⁺ OOC-R-COO ^" NET compound. The addition of CO2 may provide a additional source of carbon, for the production of the HOOC-R-COOH compound acid.

[0051 ] In another example,, the contents of growth vessel 12 may be transferred via stream 14 to a separate bioconversion vessel 16 for bioconversion of a carbohydrate sourc to the HOOC-R-C OH compound acid. An oxygen-deficient gas (e.g., CO? or the like) is. sparged In bioconversion vessel 16 to provide anaerobic Conditions that trigger production of the HOOC-R-COOH compound acid. Ammonium hydroxide is provided for pH control during bioconversion of the carbohydrate source to the HOOC-R-COOH compound acid. Due to the presence of the ammonium hydroxide, the HOOC-R-C OH compound acid produced is at least partially neutralized to the ΝΚ ^" OOC-R-COO ^* NH_f compound, leading to production of a broth that comprises the NH ₄ ^{+ "} OOC-R-C O ^* NH_f compound. The addition of C0 ₂ may provide an additional source of carbon for production of the HO C-R-COOH compound acid.

[0052] In another example, the bioconversion may be conducted at relatively low pH (e.g., 3 - 6). A base (ammonium hydroxide or ammonia) may be provided for pH control during bioconversion of the carbohydrate source to the HOOC-R-COOH compound acid. Depending on the desired pH, and dise to the presence or lack of the ammonium hydroxide, either the HOOC-R-COOH compound acid Is produced or the HOOC-R-COOH compound, acid produced Is at least partially neutralized to a N ^' Ei ^{+ "} COC-R-CO0H compound, aNfV ^" OC-R-COO ^" NRf compound, or a mixture comprising a HOOC-R-COOH compound acid, a NHV ^" OOC-R-COOH compound and/or a NH ₄ ⁺ OOC-R-COO ^' N¾ ⁺ compound. Thus, the HOOC-R-COOH compound acid produced daring bioconversion can be subsequently neutralized, optionally m an additional step, by providin either ammonia or ammonium hydroxide leading to a broth comprising the N¾ ⁺ O C-R-COO ^" NH ^" compound. As consequence, a "NiLT ^' OOC-R-COO ^* NH compound-containing fermentation broth" generally means that the fermentation broth comprises a KH ^{* "} OOC-R-COO ^' H ₄ ^' compound and .possibly any number of other components such as a NH ^" OOC-R-COOH compound and or a HOOC-R-COOH compound acid, whether added and/or produced by bioconversion or otherwise. Similarly, a ^" OQC-R-COGH-contamiug fermentation, broth" generally means that the fermentation broth comprises NIV ^" OOC-R-COOH and possibly any number of other components such as N¾f ^" OOC-R-COO ^" NH ₄ ⁺ and/or HOOC- R-COOH, whether added and/or produced by bioconversion or otherwise.

[0053] The broth resulting from the bioconversion of the fermentable carbon source {in either growth vessel 12 or bioconversion vessel 16. depending on where the bioconversion takes place), typically contains insoluble solids such as cellular biomass and other suspended material, which are transferred via stream 18 to clarification apparatus 20 before distillation. Removal of insoluble solids clarifies the broth. This reduces or prevents fouling of subsequent distillation equipment. The insoluble solids can e removed by any one of several solid-liquid separation techniques, alone or in combination, including bin not limited to centrifugation and filtration (including, but not limited to ultra-filtration, micro-filtration or depth ^' filtration). The choice of filtration technique can be made using known techniques. Soluble inorganic compounds can be removed by any number of known methods such as but not limited to ion-exchange and physical adsorption. [0054] An example of centrifugatio is a continuous disc stack centrifuge. It may be useful to add a polishing nitration ste following centrii gation such as depth filtration, which may include the use of a filter aide such as diaiomaceous earth or the like, or more preferably ultra-filtration or micro-filtration. The ultra-filtration or micro-filtration membrane can be .ceramic or polymeric, for example. One example of a polymeric membrane is SelRO MPS-U20P (pH stable ultra-filtration membrane) manufactured by Koch Membrane Systems (850 Main Street, Wilmington _* MA, USA). This is a commercially available pofyeihersulfone membrane with a 25,000 Dalton molecular weight cut-off which typically operates at pressures of 0.35 to 1 ,38 MPa (maximum pressure of 1 ,55 MPa). and at temperatures up to 50°C. Alternatively, a filtration step may be employed, such as ultrafiltration or micro -filtration alone.

[0055] The resulting clarified NH ₄ ^{+ "} OOC-R-COO ^" N¾ ⁺ compound-containing broth, substantially free of the .microbial culture and other solids, is transferred via stream 22 to distillation apparatus 24.

[0056] Water and ammonia are removed, from distillation apparatus 24 as an overhead, and at least a portion is optionally recycled via stream. 26 to bioeo version, vessel 16 (or growth- vessel 12 operated in the anaerobic mode). Distillation temperature and pressure are not critical as long s: the distillation is carried out in a way that ensures that -the distillation overhead contains water and ammonia, and the distillation bottoms comprises at least -some of the ΝΗ, OOC-R-COO ^' Ν¾ ^{' 1} compound and at least about 20 wt% water. A more preferred amount of water is at least about 30 wt% and an even more preferred amount is t least about 4 wf%. The rate of ammonia removal from the distillation- step increases with increasing temperature and also can be increased by Injecting steam (not shown) during distillation. The rate of ammonia removal during distillation may also be increased by conducting distillation under a vacuum or by sparging the distillation apparatus with a non-reactive gas such as air, nitrogen or the like,

[0057] Removal of water during the -distillation step can be enhanced by the use of an organic azeotroping agent such as toluene, xylene, cyelohexanei methyl cyeiohexane, methyl isobi!tyl ketone, heptane or the like, provided that the bottoms contains at least about 20 wt% water. If the distillation is carried out in the presence of an organic agent capable of forming an azeo rope consisting of the water and the agent, distillation produces a triphasic, bottoms that comprises an aqueous phase and an organic phase, in which c se the aqueous phase can be separated from the organic phase, and the aqueous phase used as the distiUation bottoms. Byproducts such as amides and imides of a HOOC- -COOH compound acid are substantially avoided provided the water level in the bottoms is maintained at a level of at least about 30 wt%,

[0058] A preferred temperature for the distillatio step is in the range of about 50 to about 300°C, depending on the pressure. A more preferred temperature range is about 90 to about 150 C, depending on the pressure, A distillation temperature of about 1 10 to about \4ifC, is preferred. "Distillation temperature ^' ' refers to the temperature of the bottoms (for batch distillations this may be the temperature at the time, when the last de-sired amount of overhead is taken),

[0059] Adding a water miscible organic solvent or an ammonia separating solvent facilitates deammoniation over a variety of distillation temperatures and pressures as discussed above. Such solvents include aprotic, bipolar, oxygen-containing- solvents that may be able to form passive hydrogen bonds. Examples include, but are not limited to, diglyme, triglyme, tetraglyrae, sulfoxides such as dimethyisulfoxide (DMSO) _÷ amides such as dimetnylforma ide (DMF) and dimeihylacetafnide, siilfones such as dimethylsujfone,- ^• salfolane, polyethylenegjyeoi (PEG), butoxytriglycol, ^! -methyipyroIidorse (NMF), gamma- butyrolactone (G ^' BL), ethers such as dioxane, methyl ethyl ketone (MIE ) and the like. Such solvents aid in the removal of ammonia from the ¾f ^" OOC-R-C O ^' Nf or NI¾ ⁺ OOC- R-COOH compound in ^' the clarified broth. Regardless of the distillation technique, it is important that the distillation be carried out in a way that ensures that at least, some of the Nil * OOC-R-COO ^* Nl-iy compound and at least about 20 t% water remain in the bottoms and even more advantageously at least about 30 t%.

[0060] The distillation can be performed at atmospheric, sub-atmospheric, or super- atmospheric pressures. The distillation can be a one-stage flash, multistage distillation (i.e., a multistage column distillation) or the like. The one-stage flash can be conducted in any type of flasher (e.g., a wiped film evaporator, thin, film evaporator, hermoslphon flasher, forced circulation flasher and the like). The. multistages of the distillation -column can be achieved by using trays, packing or the like. The packing can be random packing (e.g., Rasehig rings. Pall rings, Berl saddles and the like) or structured packing (e.g., Koch-Sulzer packing, inta!ox packing, eilapak and the like). The trays can be of any design (e.g., sieve trays, valve trays, bubble-cap trays and the like). The distillation can be performed with an number of theoretical stage s.

[0061], If the distillation apparatus is a column, the configuration is not particularly critical and the column can be designed using well known criteria. The column can be operated in either stripping mode, rectifying mode or fractionation mode,. Distillation can be conducted in either batch or continuous mode. In the continuous mode, the broth is fed continuously into the distillation apparatus, and the overhead and bottoms are continuously removed from the apparatus as they are formed- The distillate from distillation is an ammonia/water solution, and the disiiilaiion bottoms is a liquid, aqueous solution of a NPLf OOC-R-COOH compound and a NH/ OC-R-COO ^' NH./ compound, which may also contain other fermentation by-product salts (i.e., ammonium acetate* ammonium formate, ammonium lactate and the like) and color bodies,

[0062] The distillation bottoms can be transferred via stream 28 to cooling apparatus 30 and .cooled by conventional techniques. Cooling technique is not critical. A heat exchanger (with heat recovery) can be used. A flash vaporization cooler can be used to cool the bottoms down to about 150 C. Cooling to 0°C typically employs a refrigerated coolant such as. for example, glycol solution or, less preferably, brine. A concentration step can be ^" included prior to cooling to help increase product yield. Further, both concentration and cooling can be combined using methods known such as vacuum evaporation and heat removal using integra ted cool ing jackets and/or external heat exchangers .

[0063] We found that the presence of some of the Nil; " ^" OOC-R-COO ^* NH/ compound in the liquid bottoms facilitates cooling-induced separatio of the bottoms into a liquid portion in contact with a solid portion that at least "consists essentially" of the NH ₄ ^{+ '} OOC-R-COOH compound (meaning that the solid portion is at least substantially pure crystalline N¾ ^{+ '} OOC-R-COOH compound) by reducing the solubility of the NH/ OOC-R-COOH compound in the liquid, aqueous, NH/ ^" OC-R-C O ^* NHT ^' compound-containing bottoms. We discovered, therefore, thai the NH/ OOC-R-COOH compound can be more completely crystallized out of an aqueous solution if some of the Ni V OOC-R-COO ^" Η ^' compound is also present in that solution. A preferred concentration of a. NH/ OOC-R-COO ^" NH compound in such a solution is about 0.1 to 30 wt%. This phenomenon allows crystallization ■of the NH/ OOC-R-COOH compound (i.e., formation of the solid portion of the distillation bottoms) .at temperatures higher than those that would be required in the absence of the NH ₄ ^{+ "} OOC-R-COO ^* h ~ compound.

[0064] When about. 50% of the ammonia is removed from Nil ^" OOC-R-COO ^" N¾ ⁺ contained in an aqueous medium the dicarboxylate species establish an equilibrium mixture of Ni¾ ^{+ "} OOC-R-COO ^" N¾ ^* ¾ ⁺ OOC-R-COOH and HGOC-R-COOM within a pH range of about 4 to 6, depending on the operating temperature- and pressure. When this composition is concentrated and cooled, NH ₄ ^V OOC-R-COOH exceeds its solubility limit in water and crystallizes. When H ^{+ "} OOC-R-C H. undergoes a phase change; to the solid phase, the liquid phase equilibrium resets thereby producing more N¾ ^{+ "} OOC-R-COOH (Wlf ^" OOC- R-COO ^' l is ^T donates an ammonium ion to HOOC-R-GOOH). This causes more NH* ^" OOC-R-COOH to crystallize from solution and continues until appreciable quantities of HOOC-R-CQQH are exhausted and the pM tends to rise. As the pH rises, the liquid phase distribution favors NH ₄ ^{+ "} OOC-R-C O ^" NH ₄ ^' \ However, since H ₄ ^'r OOC-R-COO ^" NK» ⁺ is highly soluble in water, NH ^" OOG-R-COQH continues to crystallize as its solubility is lower than N¾ ^{÷ "} OOC-R-COO ^" ΝΗ in effect, the liquid phase equilibrium and the liqoid- splid equilibria of succinate species act as a "pump" for H ^{÷ "} OOC-R-COOH crystallization, thereby enabling N¾ ⁺ OOC-R-COOH crystallisation in high yield.

[0065] In addition to cooling, evaporation, or evaporative cooling described above, crystallization of Ni¾T OOC-R-COOH can be enabled arid/or facilitated by addition of an antiso!veni. In this context, antiso!vents may be solvents typically miscible with water, but cause crystallization of a ^' water soluble salt such as NH ₄ ^{+ "} OOC-R-COOH due to lower ^' solubility ^' of the salt in the solvent. Solvents with an antlsolvent effect on N.H ^" OOC-R- COOH ean be aleohois such as ethanol and propanol, ketones such as methyl ethyl ketone,, ethers such as tetrahy ^' drofuran and the like. The use of antisolvents is- known and can be used in combination with cooling and evaporation or separately.

[0066] ^' The. distillation bottoms is fed via stream 32 to separator 34 for separation of the solid portion from the liquid portion. Separation can be accomplished via pressure filtration (e.g., using Nutsche or Rosenmond type pressure filters), ^• centrifugation- and the like. The resulting solid product can be recovered as product 36 and dried, if desired, by standard methods.

|0067] After separation, it may be desirable to treat the solid portion to ensure that no liquid portion remains on the surface(s) of the solid portion. One way to minimize the amount of liquid portion that remains on the surface of the solid portion is to wash the separated solid portion with water and dry the resulting washed solid portion (not shown). A convenient way to wash th solid portion is to use a so-called "basket, centrifuge" (not shown). Suitable basket centrifuges are available from The Western States Machine Company (Hamilton, OH. USA).

[0068 The liquid portion of the distillation bottoms (i.e., the mother liquor) may contain remaining dissolved NH O C-R-COOH compound, any unconverted NH ₄ ^{* "} OOC-R-COO ^" N3¾ ⁺ compound, any fermentation byproducts such as ammonium acetate, lactate, or formate, and other minor impurities. This liquid portion can be fed via stream 38 to a downstream apparatus 40, In one instance, apparatus 40 may be a means for making a de- icer by treating In the mixture with an appropriate amount of potassium hydroxide, for example, to convert the ammonium salts to potassium salts. Ammonia generated in this reaction can be recovered for reuse in the bioconverston. vessel 14 (or growth vessel 12 operating in the anaerobic mode). The resulting mixture of .potassium salts is valuable as a de-icer and anti-icer.

[0069] The mother liquor from the solids separation step 34, can be recycled (or partiall recycled) to distillation apparatus 24 via stream 42 to further enhance recovery- of the NH*" ^{' "} OOC-R-COOH compound, as well as further convert the NH ₄ ^{÷ "} OOC-R-COO ^" M-i compound to the NH ^' OOC-R-COOH compound.

[0070] The solid portion of the cooling-Induced crystallization is substantially pure- N¾ ^{+ "} OOC-R-COOH compound and is, therefore, useful for the- known utilities of the Ν¾ ^{+ "} OOC- R-COOH compound.

[0071] HPLC can be used to detec the presence of nitrogen-containing impurities such as sttccinarnide and succi m de. The purity of a N¾ ^' OOC-R-COOH compound can be determined by elemental carbon and nitrogen analysis. An ammonia electrode ears be used to determine a crude approximation of NJV ^" OOC-R-COOH compound purity.

[0072] Depending on the. circumstances and various operating inputs, there are instances when the fermentation broth may be a clarified ΝΗ ^" OOC-R-COOH compound-contain ing fermentation broth or a clarified BOGC-R-COOH compound acid-containing fermentation broth. In those circumstances, it can be advantageous to add a N¾ ⁺ OOC-R-COOH compound, a N¾* ^" OOC-R-COO ^" MH ₄ * compound and/or a HOOC-R-COOH compound acid, ammonia and/or ammonium hydroxide to those fermentation broths to facilitate the production of substantially pure NH ₄ ⁺ OOC-R-COOH compound. For example, the operating pH of the fermentation broth ma be oriented such that the broth is a NH ^" OOC- R-COOH compound-containing broth or a HOOC-R-COOH compound acid-containing broth. A NH ^" OOC-R-COOH compound, a NH/ ^" OOC-R-COO ^" NH compound, a HOOC-R-COOH compound acid, ammonia and/or ammonium hydroxide may be optionally added to those broths to attain a broth pH less than 6 to facilitate production of the above- mentioned substantially pure NH/ ^' OOC-R-COOH compound. Also, it is possible that a NH/ OOC-R-COOH compound, a N¾ ⁺ T ⁾ OC~R~COO ^' H/ compound and/or a HOOC-R- COOH compound acid from other sources ma be added as desired. In one particular form, it is especially advantageous to recycle the Nl f ^' OOC-R-COOH compound, the NH/ OC- R-COO ^" NH/ compound and water from the liquid bottoms resulting from distillation step 34 into the fermentation broth. In referring to the NH/ ^" OOC-R-COOH compound-containing broth, such broth generally means that the fermentation broth comprises a NR./ OC-R- COOH compound and possibly any number of other components such as a NH ^" QC-R- COO ^' Tsffi/ compound and/or a HOOC-R-COOH compound acid, whether added and/or ^• produced by bioco version or otherwise.

[0073] The solid portion can be converted into a HOOC-R-COOH compound acid by removing ammonia. This can be carried out as follows. The solid portion (consisting essentially of a NH/ ^' OOC-R-COOH compound) obtained from any of the above-described conversion processes can be dissolved in water to produce an aqueous NH/ ^' OOC-R-COOH compound solution. This solution can then be distilled at a temperature and pressure sufficient to fonn an overhead that comprises water and ammonia, and a bottoms that comprises a major portion of the HOOC-R-COOH compound acid, a minor portion of the N¾ ^{+ '} OOC-R-COOH compound and water. The bottoms can be cooled to cause it to separate nto a liquid portion in contact with a solid, portion that consists essentially of the HOOC-R-C OH compound acid and is substantially free of the NH/ ^" OOC-R-COOH compound. The solid portion can be separated from the second liquid portion -and recovered ^• as substantially pure HOOC-R-COOH compound acid as determined by HPCC.

[0074] Turning to Fig. 2, we describe one of our particularly preferred processes. In. Fig. , a stream 100 of H OC-R-COO ^" NH/, which may be a stream of clarified fermentation broth which contains NH/ OOC-R-COO ^" NH* ^1" (among other things), is subjected to reactive evaporation/distillation in distillation column 102. The distillation may occur over a range of temperatures such as about 1 10 to about 145°C, preferably about 13-5 C. The pressure in the distillation column 102 can be over a broad range about 1.5 to about 4 bar, preferably about 3.5 bar. Water and ammonia are separated in distillation column 102 and form ^' an overhead- 104, The liquid bottoms 106 comprises l ^" OOC-R- COOH, at least some N¾* OOC-R-COO ^" NH and at least about 20 vvt%- water. Typically, bottoms 106 contains about 5 to about 20 t% NH ^{+ "} OOC- -COOI-L about 80 wt%- to about 95 wt% water and about ! to about.3 M% NH ⁺ OGC-R-COO ^' NB . The pH of the bottoms may be in a range of about 4.6 to about 5.6,

[0075] The bottoms 106 is streamed to a concentrator 108 which removes water via overhead stream 1 10. Concentrator 108 can operate over a range of temperatures such as about 9Q°C to about 1 10°C, preferably about I 00°C and over a range of pressures such as at about 0.9 bar to about 1 .2 bar, preferably about LI 03 bar,

[0076] Concentrator 108 produces a bottoms stream 1 12 which typically contains about 40 wt% to about 70 wt%, preferably about 55 wt% NH OOC-R-COOH. Hence, the concentrator concentrates the amount of -i ^" OOC-R-COOH typically by about 2 to about

1 I times, preferably about 4 times to about 6 times.

[0077] Bottoms stream 1 12 flows to a first crystal lizer 1 14 which operates at a temperature typically at about SO to about 70 ^a C, preferably about 60°C. A water overhead stream 1 .16 is produced by the crystai&er. Bottoms Π8 flows to a centrifuge 120 which produces a solid stream 122 which typically has a yield of ΝΗ.- OC-R-COOH -of about 95%. A -remaining liquid flow 124 is sent to a second crystal! ixer 126 which removes additional water by way of overhead -stream 128 and operates at a temperature typically at about 30 to about 50°C, preferably about -40°C. The bottoms stream 130 flows to a centrifuge 132, Centrifuge- produces a- solid stream 134 which is redissdlved with a wate stream 36 which introduces water in a temperature range typically -of about 70 to about 0°€, preferably about 90°C. That stream flows to a first mixer 138 and: produces a -first recycle flow 1-40 back to the first crystal lizer 1 14.

0078] Remaining liquid from centrifuge 132 flows via stream 141 to third crystaJlizer 142 which produces an overhead stream 144 of water. Third cry-sta!lizer 132 typically operates at a temperature of about 10 to. about 30°C,. typically about 20 ^ft C. The remaining bottoms flo 146 streams to a third centrifuge 148 and solid material produced by third centrifuge 148 flows to a second mixer 150 by way of stream 152. That solid -stream is dissolved by a second water stream 154 which introduces water typically at a temperature range of about 50 to about ?0°C, preferably about 70°C. Second mixer 150 produces a recycle stream 156 which is recycled to second crystallizer 126. Remaining material flows outwardly of the system from third centrifuge 148 by way of purge stream 158 ^' which typically represents about 5 wt% of the total Rf OOC-R-COOH contained in stream 1 12.It Is understood. that the desired crystallization temperatures in crystai!izers 1 14. 126, and 142 can be attained, by evaporation (as depicted), or by indirect contact with an external cooling medium, or a combination thereof.

Examples

[0079] The processes are illustrated by the following non-limiting representative examples. In all examples, a synthetic, aqueous H OOC-R-CO ^" ΝΆ{ compound solution such as diammonium ma late, diammonium f marate, diammonS ^' um uaeonate, diammoniun-s malonate and diammonium dodecanedionate was used in place of an actual clarified H ^" OOC-R-dQO ^' NlV compound-containing fermentation broth.

[0GS0] The use of such synthetic NHL* ^{* "} OOC-R-COO ^" H compound solutions is believed to be a good mode! for the behavior of an actual broth in our processes because of the solubility of the typical fermentation by-products found in actual broth. Typically, the major by-products produced during fermentation are salts of monocarboxylic acids such as ammonium acetate, ammonium lactate and ammonium formate. If these impurities are present during the distillation step, one would not expect them to lose ammonia and form tree acids in significant quantities until all of the H ₄ ^{* "} OOC-R-COO ^" NH ₄ ^* compound had been converted to NJ¾ ^{+ "} OOC-R-COOH ^' compound. This is because acetic acid, lactic acid and formic acid ar stronger acids than the second acid group of i!OOC-R-COOH compound acids such as malonic acid (pK.a=5.69), malic acid (pKa-5. 3),. citracon acid (pKa-6.15 to 6.2), itacontc acid (p a-5.45), muoomc acid, sebacid acid (pKa-5.450), and dodecandloic. In other words, acetate, lactate, formate and even monohydrogen succinate are weaker bases than the dianions of such HOOC-R-COOH compound acids. Furthermore, ammonium acetate, ammonium lactate and ammonium formate are significantly more soluble in water than such NH ⁺ OC~R~CQQH compounds, and each is typically present in the broth at less than 10% of the NH ^" OOC-R-COO ^" N¾ ⁺ compound concentration. In addition, even if the acids (acetic, formic and lactic acids) were formed during the distillation step, they are Tfiiscibfe with water and will not crystallize from water. This means: that the NH OC-R- COQII compound reaches saturation and crystallizes from solution (i.e.., forming the solid portion), leaving the acid impurities dissolved in the mother liquor (i.e., the liquid portion);

Example 1

[0081 J This example demonstrates ammonia evolution from aqueous NH OOC- -COO ^" NHf compounds such as diammonium maiaie, diammonium itaconate, diammonuim raalonate and diammonium dodeeanedionate.

[00821 The outer necks of a three neck 1 -L round bottom flask were fitted with a thermometer and a stopper. The center neck was fitted with a five tray 1" O! ershaw section. The Oldershaw section was topped with a. distillation head. An ice cooled 500 mL round bottom flask was used as the receiver for the distillation head. The 3 -L round bottom flask was charged with distilled water, the HOOC-R-COOH compound acid indicated in Table S and concentrated ammonium hydroxide solution. The contents were stirred with a magnetic stirrer to dissolve ail the solids. After the solids dissolved, the contents were heated with the heating mantle to distill 3 ^' 50g of distillate. The distillate was collected in the ice cooled .500 mL round bottom flask. The pot temperature was recorded as the last drop of distillate was collected. The pot contents were allowed to cool to room temperature and the weight of the residue and. weight .of the distillate were recorded. The ammonia content of the distillate was then -determined via titration. The results were recorded in Table 1 ,

Table 1

Run 1 4 5

Name of Acid Malic itaconic M ^' alorsic DDA Fumaric Citric

Wt Acid Charged (g) 13.4 13.01 10.44 23.04 1 ! .62 19.22

Moles Acid Charged 0, 1 0.1 0.1 0.1 0.1 0.1

Wt 28¾ NH _S Solution Charged (g) 12. i i 12.1 1 2.18 ! 2.1 12.1 18,27

Moles NH Charged 0,2 0.2 0.2 0.2 0.2 0.3

W Water Charged (g) 800.21 800.54 800, 1 S0G.1 800.79 800.24

Wt Distillate (g) 350,78 350.14 350.5 350 350.61 350 85

Wt Residue (g) 461.99 467 467 465 468.49 478.93

%Mass Accountability 98.5 99 99. 97.6 99.3 99

Wt% NH-, in distillate (titration) 0.07 0.1 1 0.29 0.13 0.06 0,2

Moles Nf¾ in distillate 0.0 is 0.023 0.06 0.027 0,012 0.041 Total !¾: removed in Distillate 1 1.3 30 13 6 13.7

%First NH ₃ removed in Distillate 14.4 22.6 60 26 12 41. J

Di.NiV θίίθΝΗ* 86/14 77/23 40/60 74/26 88/12

Final Pot Temp 100 100 1 Q0 100 100 100

Micromoles of NHj ^' g distillate 41 63 171 77 34 1 !7

Initial Wt ammonium salt 2 2.1 i .7 3.2 1.9 3 pKa< 3.4 3.85 2.83 unk 3.03 5.14 p ^' a ₃ 5.1 1 5.45 5.69 unk 4 _* " " 4.77 p .a ₃ NA NA NA NA NA 6.39 [0083] Although our proc sses have been described tn connection: with specific steps and forms thereofl it will be appreciated that a wide variety of equivalents may be substituted for the specified elements and steps described herein without departing from the spirit and scope of ibis disclosure as described in. the appended claims.