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WO1997010350A1 - Procede, appareil et souche de micro-organismes pour fabriquer de l'acide citrique - Google Patents

Procede, appareil et souche de micro-organismes pour fabriquer de l'acide citrique Download PDF

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Publication number
WO1997010350A1
WO1997010350A1 PCT/SK1996/000014 SK9600014W WO9710350A1 WO 1997010350 A1 WO1997010350 A1 WO 1997010350A1 SK 9600014 W SK9600014 W SK 9600014W WO 9710350 A1 WO9710350 A1 WO 9710350A1
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WIPO (PCT)
Prior art keywords
citric acid
fermentation
strain
separation
crystallisation
Prior art date
Application number
PCT/SK1996/000014
Other languages
English (en)
Inventor
Martin Minarik
Dus^¿an S^¿KVARENINA
Peter MICHALÍK
Vladimír SITKEY
Viliam VIS^¿ACKY
Original Assignee
Likospol S.R.O.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from SK113195A external-priority patent/SK113195A3/sk
Priority claimed from SK156-96A external-priority patent/SK281100B6/sk
Priority claimed from GBGB9607868.8A external-priority patent/GB9607868D0/en
Application filed by Likospol S.R.O. filed Critical Likospol S.R.O.
Priority to AU70061/96A priority Critical patent/AU7006196A/en
Publication of WO1997010350A1 publication Critical patent/WO1997010350A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/10—Separation or concentration of fermentation products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/12—Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/48—Tricarboxylic acids, e.g. citric acid

Definitions

  • the present invention relates to a process for producing purified citric acid or citric acid concentrate which comprises producing by fermentation a raw aqueous citric acid solution and subjecting it to a preliminary physical separation of citric acid from the bulk of its major, mainly macromolecular contaminants and optional subsequent purification steps.
  • the present invention also relates to a new strain of Aspergillus niger, capable of fermenting carbohydrate substrates to citric acid and forming a fermentation broth suitable for the above process.
  • the invention further relates to an apparatus for carrying out the process, comprising a fermentation stage and optional separation means capable of producing a raw citric acid solution and a downstream preliminary separation stage wherein citric acid is separated from the bulk of its major mainly macromolecular contaminants.
  • carbohydrate substrates are fermented to raw aqueous citric acid solutions using a variety of micro-organisms, including yeasts, but in particular Aspergillus niger conidia. This is followed by sieve and press filtration mainly to remove the mycelium. This is followed by the step of preliminary separation of citric acid from the bulk of its major, mainly macromolecular contaminants in particular proteins.
  • Other bothersome impurities which have to be removed in the more classical process in order to arrive at a citric acid product of high grade (e.g. pharmaceutical grade such as BP 93) are residual sugar contents and acids. Oxalic acid contamination is particularly bothersome because of its toxicity.
  • a disadvantage of this process is that membranes having a separation cut as low as 1000 dalton offer a high resistance to the flow of the permeate so that for attaining reasonable throughput rates it is necessary to employ excessively large membrane areas and energy inputs for pumping the permeate through the membranes. Such membranes also clog after relatively small volumes of throughput.
  • German patent application 2 450 670 discloses a similar process for separating citrates and citric acid and/or isocitric acid from organic and inorganic contaminants using a membrane selective towards citric acid and citrates.
  • Nanofiltration denotes a form of ultrafiltration, wherein the substances being separated are of very low molecular weight.
  • PCT/US 95/12160 filed and published after the earliest priority date of the present application relates to a process for the purification of citric acid which includes separating citric acid from residual sugars of the broth by nanofiltration. According to the disclosure the broth leaving the fermenter can contain 0.2% (w/w) unfermented fructose. The separation of citric acid from sugars by nanofiltration, is relatively incomplete.
  • citric acid A relatively large proportion of the citric acid remains in the retentate and a substantial proportion of the sugars remains in the permeate, so that (besides ion exchange and activated carbon absorption) a mere repeated crystallisation of the permeate may not suffice to produce highest quality crystalline citric acid.
  • the fermentation broth must inter alia have a low content of or be substantially free of noxious organic acids, in particular D
  • citric acid being of a grade as required especially in the food industry, pharmaceutical industry, beverage industry and other industries.
  • a raw aqueous citric acid solution is produced wherein contaminants include not more than 2 000 mg/1 of sugars and a ratio of citric acid to sugars exceeding 100 : 2; b) the said preliminary physical separation of the citric acid from the bulk of its mainly macromolecular contaminants is performed using a separation device having a separation cut in the range between 5000 and 20 000 dalton.
  • the invention is based on the concept of initially preparing a fermentation broth having a low residual sugar content, a high ratio of citric acid to residual sugar and generally a low content of low molecular weight organic contaminants, so that by physical separation according to molecular weight employing a separating cut at a relatively high molecular weight of 5000 - 20 000 dalton a solution is obtained from which a high purity citric acid can be obtained by straight forward crystallisation of the free acid.
  • step b) is preferably performed by ultrafiltration.
  • step b) is performed at a pH of below about 2, in particular 1.6 or less, e.g. 1.4 - 1.5, using an ultrafiltration device, i.e. a separation membrane, sufficiently stable at such pH and, by virtue of its aforesaid separation cut, capable of retaining macromolecular substances having a molecular diameter greater than a value between 5000 and 20 000, preferably greater than 10 000, whilst allowing substances of lower molecular weight, e.g. any residual non-fermented sugars to permeate.
  • an ultrafiltration device i.e. a separation membrane, sufficiently stable at such pH and, by virtue of its aforesaid separation cut, capable of retaining macromolecular substances having a molecular diameter greater than a value between 5000 and 20 000, preferably greater than 10 000, whilst allowing substances of lower molecular weight, e.g. any residual non-fermented sugars to permeate.
  • the ultrafiltration device has the said separation cut when used at a temperature of 20 to 45 °C and at a pressure not exceeding 2 MPa. More particularly the process is so performed that a filtrate of the ultrafiltration is obtained with a citric acid content exceeding 100 g/1, more particularly exceeding 130 g 1, preferably up to about 170 g/1, a sugar content of not more than 2000 mg/1 and preferably considerably lower and a residual content of readily carbonisable substances (RCS) not exceeding 15 U, preferably not exceeding 13 U, and that this solution is subjected to a step of direct citric acid crystallisation.
  • RCS readily carbonisable substances
  • RCS readily carbonisable substances
  • RCS has to be in the range of 9-15 U.
  • RCS values in the range of 9-13 U have been readily attained by the present invention.
  • the RCS content should not exceed 170 U.
  • An important preferred feature of the invention therefore resides in that the raw aqueous citric acid solution is produced by the fermentation of a carbohydrate substrate until its sugar level is reduced to not more than 1000 mg/1 and its citric acid content exceeds 130 g/1, preferably 150 g/1, being for example as high as about 165 g/1.
  • the fermentation itself is performed so effectively that the sugar level is reduced to not more than 1000 mg/1, preferably to less than 800 mg/1, e.g. 600 mg 1 (and even as low as 500 mg/1), in spite of the aforesaid relatively high citric acid content, being achieved and the resultant comparatively high acidity of the fermentation liquor.
  • the fermentation is commenced at a pH not exceeding pH 4 preferably not exceeding pH 3 and more preferably at a pH of 2.8 or less.
  • a pH not exceeding pH 4 preferably not exceeding pH 3 and more preferably at a pH of 2.8 or less.
  • the invention teaches the combined employment of particularly suitable strains of Aspergillus niger in combination with a substrate well adapted to such strains.
  • the substrate is a starch hydrolysate, e.g. starch hydrolysate produced with bacterial or fungal amylase or glucoamylase at 45 - 50° C or sucrose solution, preferably a sucrose solution which may optionally be of brown sugar quality, and the Aspergillus niger conidia used is a strain having essentially the aforesaid performance characteristics.
  • starch hydrolysate e.g. starch hydrolysate produced with bacterial or fungal amylase or glucoamylase at 45 - 50° C or sucrose solution, preferably a sucrose solution which may optionally be of brown sugar quality, and the Aspergillus niger conidia used is a strain having essentially the aforesaid performance characteristics.
  • the strains of micro organism preferred for use in accordance with the invention derive nutritional benefits from the presence in the substrate of the "impurities" present in grades of sugar which have not been fully refined.
  • the process can be performed with a hydrolysed starch substrate, wherein the starch may be derived from a large variety of commercial starch sources.
  • the hydrolysed starch substrate may be derived from any suitable commercial source of starch such as maize (corn), wheat, rice or other cereals, potatoes, sago, yam, or cassava.
  • a preferred strain of Aspergillus niger is a strain known as CCM 8210, or a strain having essentially the same performance characteristics.
  • CCM 8210 was deposited at Czech Culture Collection of Micro-organisms under that number on 15 November, 1995.
  • the strain is said No CCM 8210 as such.
  • Inventiveness is claimed for that strain as such, and this will be dealt with more fully below.
  • strain has the property of being able to start the fermentation at a surprisingly low pH, thereby substantially suppressing the formation of oxalic acid and other noxious acids.
  • This strain also offers the advantage that it can be used for fermenting starch-containing sugar juices e.g. derived from sugar cane having a substantial starch content.
  • the invention is not restricted to that strain but covers the use of other strains if capable of reducing the particular substrate to the required parameters. More particularly, if the fermentation is performed on a sugar substrate, more particularly a refined or partly refined sucrose solution, a commercial strain known as B-64-5 may be used.
  • RCS has to be in the range of 9 - 15 U.
  • RCS values in the range of 9-13 U have been readily attained by the present invention.
  • a further preferred aspect of the invention provides that after said step of direct crystallisation an amount of mother liquor from the direct crystallisation equivalent to from about 5 to 15 % of the total citric acid formed by the fermentation is withdrawn and passed to separate processing.
  • the amount of mother liquor withdrawn is equivalent to 7 to 13% of the total citric acid, more preferably about 10% of the total citric acid.
  • the mother liquor withdrawn is subjected to steps for the recovery of citric acid: a) in purified form by calcium citrate precipitation, or b) as sodium citrate, or c) as liquid technical grade citric acid, or more than one of these.
  • the solution obtained from the preliminary separation is subjected to purification by absorption and/or ion-exchange, preferably both.
  • the crystallisation product of said direct crystallisation steps is subjected to at least one further purification step involving direct recrystallisation of citric acid in its non-neutralised form.
  • the solution obtained from the preliminary separation is subjected to purification by absorption and/or ion-exchange. Also where as described above a portion of the mother liquor from this step of direct crystallisation is withdrawn and passed to separate processing, the balance of the mother liquor is recycled to the said crystallisation.
  • the crystallisation product of said crystallisation step is subjected to at least one further purification step involving direct recrystallisation.
  • the invention also provides an apparatus for carrying out the process, including the features as set out in the opening paragraph, comprising, in accordance with the invention the novel feature that the downstream preliminary separation stage is a physical separation device and wherein more particularly the physical separation device is an ultrafiltration device, having a separation cut in the range 5 000 - 20 000 dalton, more particularly 10 000 dalton, more particularly when used at a temperature of 20 to 45 °C and at a pressure not exceeding 2 MPa.
  • the apparatus more particularly comprises a direct crystallisation stage for crystallising citric acid and separating it from its mother liquor, followed downstream by at least one further purification stage and optionally a passage for withdrawing part of the said mother liquor and feeding said part to separate and different citric acid recovery means and a passage for recycling remaining mother liquor to the said direct crystallisation stage.
  • a membrane device having those characteristics and stable at pH 1.2 has been developed according to the applicant's specification and is manufactured by Unipektin A.G., Zurich, Switzerland. However, any other suitable ultrafiltration device satisfying the aforesaid requirements regarding stability in acid medium and ability to retain macromolecular impurities, may be employed. From the preferred ultrafiltration step, using an ultrafiltration medium as aforesaid, a permeate may be obtained containing not more than 0.03 g/1 of proteins.
  • said at least one further purification stage includes a further direct crystallisation stage, wherein citric acid is recrystallised.
  • the aforesaid separate and different citric acid recovery means (for the withdrawn part of the said mother liquor) includes a calcium citrate precipitation stage or a sodium citrate recovery stage or a liquid technical citrate recovery stage or a plurality of these.
  • the new strain has the ability to continue the fermentation until the residual sugar level of the substrate has dropped to less than 800 mg/1, e.g. 600 mg/1 and to start said fermenting at a pH at least as low as pH 2.9, preferably at a pH at least as low as pH 2.8.
  • the new strain has the ability to continue fermentation until the pH of the substrate has dropped to at least as low as pH 1.3, preferably 1.2.
  • the preferred embodiment of the strain has the ability to ferment a starch or sugar substrate, optionally of brown sugar quality, to a citric acid solution capable of being further purified for citric acid recovery by ultrafiltration without prior calcium citrate precipitation.
  • the preferred embodiment of the strain is capable of producing a fermentation broth wherein the ratio of citric acid to residual sugar exceeds 187.5 : 1 and may be as high as 275 : 1 to about 300 : 1.
  • the preferred embodiments have the ability to ferment saccharose, glucose, maltose, starch hydrolysates, lignified starches and cereal flours such as wheat flower to citric acid.
  • the preferred strain substantially has the characteristics of Aspergillus niger strain No CCM 8210. That latter strain No CCM 8210 has been deposited at Czech Culture Collection of Micro-organisms, Ceska sbirka mikroorganism ⁇ Masarykovy univerzity, Tvrdeho 14, 602 00 BRNO, under accession No CCM 8210 on 15 November, 1995. More preferably, the strain used is, in fact, the said strain No CCM 8210 as such. The strain was developed from an earlier strain obtained using protoplasts isolation from selected A. niger.
  • the protoplasts were isolated from the hyphae with helical gastric juice in stabilised aqueous solution (0.7 MNaCl -f- glucose) and addition of calcium ions. The protoplasts were separated by filtration and washed with water. Suspensions of the o protoplasts were radiated with UV (5.9 J/m /sec) for 5 minutes. Protoplast fusion was performed with wild strains selected for high growth and production ability, performed in a polyethylene glycol-calciumchloride system.
  • This A. niger strain was subjected to further mutation by a combination of UV radiation and chemical mutagens, i.e. 5-bromouracide, 2-aminopurine, diethylsulphate, ethylethane sulphonates and combinations of these. From this strain CCM 8210 was arrived at by selection of the high producing mutants, cultivated on sugar media. Bromcresol green was used as an indicator of acids formation. Selected monospore cultures were cultivated on media having high citric acid concentration in order to select strains tolerant to citric acid and low pH.
  • chemical mutagens i.e. 5-bromouracide, 2-aminopurine, diethylsulphate, ethylethane sulphonates and combinations of these.
  • CCM 8210 was arrived at by selection of the high producing mutants, cultivated on sugar media. Bromcresol green was used as an indicator of acids formation. Selected monospore cultures were cultivated on media having high citric acid concentration in order to select strains
  • the invention includes the use of a strain as aforesaid for the production from a carbohydrate fermentation broth of a citric acid fermentation liquor purifiable by purely physical means, in particular ultra filtration to a solution from which substantially pure citric acid can be recovered by direct crystallisation.
  • the invention also provides a process and an apparatus as set out in the opening paragraph, wherein the fermentation is carried out with a strain as described above.
  • the preliminary separation is performed by direct physical separation of the dissolved citric acid from contaminants of larger molecular size or weight.
  • aqueous citric acid solution containing in practice 10 to 20% by weight citric acid, 0.1-1.0% by weight proteins and percentages of reducing sugars as low as described above.
  • the strain is ideally suitable for and employed in a process as more fully described above.
  • the above strain to be used in the preferred embodiment was deposited at the Czech Culture Collection of Micro-organisms on 15 November, 1995.
  • One of its main characteristics is the ability to metabolise mono- and disaccharides and their mixtures and in particular also those resulting from the hydrolysis of pure starch and of starch-based materials by high conversion into citric acid by submerged culture.
  • the strain CCM 8210 is capable of commencing fermentation at low pH, namely as low as pH 2.8 or less, preferably pH 2.5 - 2.7 or less where the formation of such undesirable organic acids, notably oxalic acid, is suppressed.
  • the strain has a higher conversion speed than conventional strains, namely of 1.3 g citric acid per 1 litre of fermentation broth per 1 hour.
  • the strain produces 90-93 kg of citric acid from 100 kg of sugar.
  • the strain is grown on potato glucose agar slant. After 7 days of cultivation, colonies are formed up to 6.0 cm in diameter with total sporuiation. The bottom side of the culture is cream yellow. The spores are brown-black and have a size of 5 - 6 ⁇ m.
  • the viability of the spores by cultivation on the abovementioned agar slant is 3 months in terms of preserved production properties. In lyophilised form the viability is preserved for 3 to 5 years.
  • the block denoted as 0 is normally not part of the apparatus proper, that is to say, it is usually operated separately in a microbiological laboratory facility where spores of a selected A niger strain are activated under sterile conditions in advance of being fed into the seed fermenter 1.
  • the seed fermenter 1 includes an agitator operated with sterile air. It is followed downstream by a main fermenter 2, likewise equipped with agitating and sterile aeration means and operating under elevated pressure.
  • the seed fermenter 1 and the main fermenter 2 are preferably designed to operate batchwise, and for that reason a commercial plant will normally have a plurality of fermenters 1 and 2 in parallel, each fermenter being operated in a stage of the fermentation process different from the others so that when one fermenter approaches the end of the fermentation in that section of the process, another one is at a less advanced stage, thus permitting the remainder of the process to be operated continuously or semicontinuously.
  • the fermenters 1 and 2 are supplied with sterilised water through sterilising filtration device 3 and with sterile nutrients solutions through a sterilising filtration device 4.
  • anti-foaming agent from an anti-foam agent source 5 and with substrate e.g. sucrose solution through a continuous sterilisation device 6 having a steam supply 8, a cooling water inlet 7 and a cooling water outlet 9.
  • the main fermenter(s) 2 is/are followed downstream by a sweep sieve 10 and a multi-leaf pressure filtration plant 11 cooled with cooling water 13 and from where mycelium is collected at 12.
  • the mycelium separation 12 preferably includes a belt filter on which the mycelium is freed of residual citric acid by rinsing and pressing in countercurrent, the citric acid rinsing liquor being recycled to the sieve and/or filtration stages 10, 11.
  • An optional filtration bypass line 14 is shown leading from upstream of the pressure filtration stage 11 to downstream thereof.
  • the main pressure filtration stage 11 is followed downstream by a prefilter stage 15 for fine filtration and clarification prior to the stage constituting one of the main features of one aspect of the present invention and serving for the preliminary separation of citric acid from major, mainly macromolecular contaminants, being adapted for such separation to be performed by direct physical separation of the dissolved citric acid from contaminants of larger molecular size or weight.
  • this stage denoted as 16 takes the form of an ultrafiltration device capable of retaining macromolecular substances, in particular proteinaceous byproducts of the fermentation, having a molecular weight greater than 10 000 Dalton.
  • the ultrafiltration device In order to be able to perform this task on the clarified fermentation broth containing the citric acid substantially in a non-neutralised condition, the ultrafiltration device must be stable in such acidic medium, more particularly at a pH of below 2 and preferably at a pH at least as low as 1.2.
  • a suitable ultrafiltration membrane, satisfying these conditions has been developed in accordance with specifications designed by the present applicant and is available from Unipectin A.G. in Ziirich. The use of such ultrafiltration membrane in a process as set out herein is considered to be within the scope of the present invention.
  • a battery of ultrafiltration units in series each comprising a circulation pump, an ultrafilter and a cooler, each successive module being connected to receive the overflow from the preceding module.
  • the modules are connected to a supply of tap water 18 for backwashing from time to time.
  • the filter cake consisting mainly of protein concentrate is collected at 17.
  • a duct 19 for collecting the permeate from the ultrafiltration device feeds into a set of carbon columns 21 charged with activated charcoal for the decolourisation of the citric acid solution and the absorption of any absorbable impurities.
  • three carbon columns at a time are connected in series whilst a fourth column is subjected to regeneration or standby.
  • Downstream of the carbon columns a battery of cation exchange columns 22 is provided connected to a source of demineralised water 23 and a source of regeneration solution 24 and an eluate discharge line 25.
  • two cation exchanger columns are connected in series at any time and a third column is not connected whilst being regenerated or on standby.
  • the cation exchanger battery 22 is followed downstream by an anion-exchanger column battery 26 which again is connected to a source of demineralised water 27 and a source of regenerating solution 28 and a discharge line 29 for eluate. Again two columns are connected in series at any one time whilst a third column not connected is subjected to regeneration or is on standby.
  • the ion-exchanger columns feed into a first direct crystallisation stage comprising an evaporator 30 heated with steam 31 and from where vapour 32 is discharged.
  • the evaporator is followed downstream by a first crystalliser 40 and a first centrifuge 42.
  • the first crystalliser 40 includes a steam feed duct 41 for operating a steam ejector (not shown) which generates a vacuum in the crystalliser 40.
  • the first centrifuge 42 includes a supply 43 for demineralised water and an outlet for rinsing water 47 as well as an outlet 44 for mother liquor from the centrifugation. This outlet 44 is divided into a branch 46 which returns mother liquor to the first crystalliser and another branch 45 which bleeds off a portion of the mother liquor to a separate purification stage which will be described more fully further below.
  • the first centrifuge 42 feeds the crystals from the first direct crystallisation stage into a diluting vessel 48 supplied with demineralised water 49 and followed downstream by a second crystallisation stage comprising a second crystalliser 50 again supplied with steam 51 for a steam ejector (not shown) and including a vapour discharge 52 and a second centrifuge 53 with a supply of demineralised water 54 a mother liquor outlet 55 and a rinsing water outlet 58.
  • the mother liquor outlet 55 branches into a duct 56 which returns part of the mother liquor to the second crystalliser 50 and a further duct 57 which returns the balance of the mother liquor to the first crystalliser 40.
  • the solids collected in the second centrifuge pass into a drier 59 supplied with drying air 60 and a recycling duct 61 for recycling oversize (agglomerated) particles back to the diluting vessel 48.
  • the drying section 59 is followed downstream by a sorting section 62 where classification takes place by sieving into desired fractions. These fractions are passed to the packing section 63.
  • this is basically a calcium citrate precipitation purification section and may be designed in a substantially conventional manner except for the optional omission of protein precipitation means.
  • the section comprises a precipitation section 70 with feed means for lime milk 71, Ca(OH)2-
  • the precipitation stage feeds into a filtration stage 72 which includes a withdrawal line for filtrate (mud) 73.
  • the filtration stage 72 includes a belt filter divided into four sections of which the last one is used for cleaning the belt. The remaining sections serve for separating the precipitate and for washing.
  • the precipitated calcium citrate is fed into a decomposition section 74 having a supply of sulphuric acid 75.
  • a filtration section 76 Downstream of the decomposition section 74 there follows a filtration section 76, once again including a belt filter having four sections as aforesaid for separating the solution of citric acid from precipitated gypsum 78.
  • the filtrate from section 76 passes through a final filtration section 77 where further gypsum 79 is withdrawn.
  • the clarified filtrate 80 may be converted into sodium citrate in a manner known per se 81 or it may be concentrated and marketed as a liquid concentrate of citric acid 82 or it may be returned to duct 20 and recycled onto the activated carbon columns 21.
  • This part of the fermentation process is performed on a laboratory scale in the microbiological laboratory in a sterile activating medium prepared in the usual manner from food grade sucrose (150 g/1), and citric acid monohydrate (2.0 g/1) in tap water.
  • the pH is 2.5 and. a temperature of 34 ⁇ 1°C is maintained.
  • the A. niger of the selected strain, e.g. CCM 8210, are activated for about six hours in a biological incubator until the desired degree of swelling of the spores is observed microscopically.
  • Carbohydrate solutions and nutrient solutions are prepared having the compositions described in the working examples.
  • the carbohydrate solution is first heated up to 100° C, then further sterilised at 140° C and cooled to 50° C before being fed into the respective fermenters 1 and 2.
  • the nutrient solutions are brought to the desired concentrations by dilution with drinking water and sterilised by sterilising filtration (3, 4).
  • Vegetative inoculum preparation .1.
  • the sterile hydrocarbon solution and, nutrients solutions and any required sterile filtered drinking water are dosed into the seed fermenters.
  • the pH is adjusted to pH 2.5 with concentrated sulphuric acid.
  • the sterile broth is inoculated with the activated spore suspension.
  • small amounts of sterile potassium ferrocyanide solution are added to stimulate such biosynthetic activity of the culture which leads to an optimum citric acid production rate. Cultivation of the vegetative inoculum proceeds for about 48 hours with optimum agitation and aeration with sterile filtered air, sterile antifoaming agent being added when needed for foam control.
  • the broth is fully saturated with oxygen by aeration with sterile air for 30 minutes.
  • the temperature is adjusted to 32° C and vegetative inoculum is admitted from the seed fermenters using elevated air pressure.
  • the main fermentation proceeds with controlled agitation and aeration under a slightly elevated pressure of 0.3-0.5 bar, pH to drop naturally, a p ⁇ 2 of not less than lmg ⁇ 2/l and a temperature of 32° C.
  • the substrate composition, biomass and product concentration and carbon source conversion are constantly monitored. Microscopic observations are also made. Over the fermentation period of about 144 hours the substrate concentration is continuously decreased, until at the end a sugar concentration of less than 0.8 g/1 and a citric acid concentration of at least 130 g/1 is reached. In practice the sugar content may drop to as low as 0.6 g/1 and the citric acid (monohydrate) may reach 165 g/1.
  • the content of the respective fermenter is discharged into citric acid collecting drums via the sweep sieve 10 by means of the excess pressure in the fermenter 2.
  • the citric acid is then passed to pressure filtration through one of two leaf filter presses, while the second such press is cleaned of mycelium and any other solid debris and regenerated with water.
  • the filtrate is fed through the prefilter 14 into the cooled ultrafiltration plant 16.
  • the pressure drop through the ultrafiltration membrane is monitored to determine the time for cleaning the membrane of protein while switching to another ultrafiltration unit.
  • the protein residue is freed of citric acid by rinsing with water in countercurrent on a belt filter. Purifying citric acid solution (21, 22, 26)
  • the ultrafiltrate is decolorised on the battery of carbon columns 21 of which at any one time one column is regenerated with caustic soda (NaOH), reactivated with dilute sulphuric acid and washed with demineralised water.
  • CaOH caustic soda
  • the citric acid solution having passed through the carbon columns is decationised on two cation exchanger columns 21 in series while a third column is being regenerated and held on standby.
  • the potassium content of the eluate of the first column serves to indicate cation saturation of the column. Regeneration proceeds with dilute sulphuric acid, a portion of which is recycled as preregenerating liquor.
  • the decationised citric acid solution is then deanionised by passing through two anion exchanger columns in series, while a third column is being regenerated with caustic soda and then held on standby.
  • the sulphate anions content of the eluate serves as indicator for the column saturation.
  • the regeneration eluates from the carbon and ion exchange columns are collected for the recovery of offgrade citric acid solutions.
  • the deionised dilute citric acid solution enters the evaporator 30 where the solution is concentrated by heating with steam.
  • Concentration of the citric acid takes place by multiple stage evaporation until citric acid crystallises and is passed through a crystal overflow drum and a crystal suspension vessel via a hydrocyclone to be separated in the first centrifuge 42.
  • the crystals collected in the first centrifuge 42 are redissolved in a dissolving vessel. Mother liquor from the centrifuge 42 is mostly recycled into the first crystalliser 40. A small portion (about 10%) is withdrawn (45) as offgrade citric acid solution or for separate processing.
  • the citric acid from 42 redissolved at 48 is once again concentrated with heat and vacuum and passed, again through a crystal overflow drum and a crystal suspension vessel via a hydrocyclone (not shown), this time into the second crystalliser vessel 50 and from there into the second centrifuge 53. Centrifuged crystals are fed via a wet feeder (not shown) into the dryer 59. Mother liquor from the centrifuge 53 is partly recycled (55) to the second crystalliser 50 and partly (57) to the first crystalliser 40.
  • the dryer 59 is a wet conveyor dryer where the crystals are dried by hot air and then passed into a dry crystal separator 62 equipped with a dust separator and a classifier. There the crystals are sorted by sieving into three fractions. Oversize particles, e.g. over 2.0 mm are returned to the first crystalliser 40 to be redissolved and passed once again through the two crystallisation stages.
  • vented vapours are subjected to scrubbing to recover citric acid entrained in those vapours.
  • Offgrade citric acid solutions in particular the small portion of mother liquor (45) withdrawn from the first centrifuge 42 may now be treated, e.g. substantially in a conventional manner, by precipitation of calcium citrate with lime milk (70, 71).
  • the lime milk is continuously dosed into the precipitation reactor 70 and heated with steam to 85° C to form a suspension of calcium citrate which is dropped onto a band filter 72, divided into four sections of which the last one is used for cleaning the band.
  • water is separated from the suspension.
  • the filter cake is then washed with hot water in countercurrent through successive sections, the water being finally sucked off under vacuum.
  • the dried filter cake drops into a decomposition vessel 74 where it is decomposed with sulphuric acid to form citric acid and precipitated gypsum.
  • the solid gypsum is filtered off on a second band filter 76 similar to band filter 72.
  • the gypsum filter cake is washed in countercurrent with water.
  • the filter cake is washed with hot water. The washing liquors from the second and third sections are recycled for washing of the cake.
  • the filtrate is passed through a final filtration 77 before (80) being recycled (20) to the top of carbon columns 19.
  • the filtrate may be concentrated and marketed as a commercial grade citric acid solution.
  • spore conserve is a mixture of spores and carbon in a ratio of 1 : 1 - 2, stored at 12 - 20°C and a relative humidity of not more than 70%.
  • the surface of agar in aluminium dishes is inoculated from the starting culture.
  • the dishes are put into a cultivation car, first at 32°C for 8.5 days, when the relative humidity is decreased from 80 - 90% down to 60 - 70%. During that period the number of culture dishes increases from 4 - 6 initially to about 40 dishes. As from the 10th day the temperature of the car is kept at 20 - 25° C.
  • the dried spores are mixed with agitation with active carbon in a ratio of 1 : 1. This mixture is used for the fermentation process.
  • 100 litre of the sterile cultivation medium, pH 2.7 - 2.8, is inoculated with 2 - 3 vol% of 40 - 48 hour inoculum of A. niger CCM 8210 and is cultivated at 32 - 33° C for 155 - 160 hours at a tangential impeller tip speed up to 5.2 m.s " 1 and an aeration rate of 0.2 - 0.35 WM (volume of air per volume of bioreactor per minute).
  • the final concentration of impurities in the fermentation broth after biomass and prefiltration is in the range 2.0 - 3.0 g/1.
  • This broth is treated as above described with reference to the drawing.
  • concentration of impurities smaller than 10 000 dalton in the permeate of the ultrafiltration is in the range 0.1 - 0.15 g/1, being mostly pigments (aspergillin), peptides (products of protein hydrolysis) and saccharides (8 glucose units and higher).
  • the residual unfermented sugar content is 650 mg/1. Oxalic acid was completely absent.
  • the citric acid yield was 165g.l " .
  • the sterile medium is inoculated at pH 2.8 - 3.0 in a 15m fermenter with 2 - 3 vol% of 40 - 48 hour A.niger CCM 8210 inoculum and is cultivated at 32% and an aeration rate of 0.25 - 0.35 WM for 155 - 160 hours. Further treatment as in Example 1. Yield of citric acid 165g.l " . Oxalic acid was completely absent. Residual sugar 0,5 g/1.
  • composition of fermentation liquor used analogously to Example 1 or 2:
  • *could be prepared as liquefied starch, maltose or glucose syrup in various concentrations.
  • composition of fermentation liquor used analogously to Example 1 or 2:
  • sucrose 180 kg.m ammonium nitrate 2.5 kg.m potassium dihydrogenphosphate 0.25 kg.m
  • zinc sulphate 0.02 kg.m copper sulphate 0.025 kg.m
  • the permeate (9 000 1) is subjected to decolourisation on active charcoal columns, deionisation on cation exchangers and anion exchangers and concentration in a vacuum evaporator to 850 g/1.
  • the concentrate is subjected to diafiltration resulting in:
  • This strain (Commercially available: 1. Lesniak, W.: Dobor podloza fermentacyjnego dla nowo wyizolowanego wysokoaktywnego mutanta Aspergillus niger. Prace Naukowe AE Wroclaw, Technologia, 1975, 69, 93. 2. Lesniak, W. : Selekcja wysokoaktywnych szczepow Aspergillus niger dla fermentacji wglebnej kwasu cytynowego. Prace naukowe AE Wroclaw, Technologia, 1977, 188, 21.) is particularly suitable for the citric acid fermentation of refined sucrose.
  • the sucrose solution should have a concentration of approximately 50% w/w.
  • the nutrients are employed in 10% ww solutions to provide the following concentrations in the broth: - potassium hydrogen phosphate 0.2 g/1 ammonium nitrate 2.0 g/1 magnesium sulphate 0.2 g/1
  • citric acid liquors obtained by the above Examples 3, 4 and 6 are subjected to the purification process as described above.

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Abstract

L'invention concerne un procédé pour produire un acide citrique très pur. Le milieu de culture enrichi en acide citrique est obtenu par fermentation (0, 1, 2) à partir d'un substrat d'hydrate de carbone (sucrose, saccharose, glucose, dextrose, amidon hydrolysé) en utilisant une souche sélectionnée Aspergillus niger. La fermentation est démarrée à un pH bas, par exemple 2,8, pour éviter la formation d'acides toxiques (en particulier de l'acide oxalique). La fermentation est poursuivie jusqu'à un niveau résiduel de sucre très bas, en dessous de 2000 mg/l, par exemple de 600 mg/l et des rapports acide citrique/sucre résiduel très élevés, par exemple 275 : 1. Après une filtration grossière (10, 11, 15), l'acide citrique est soumis à une ultrafiltration (16), en utilisant une membrane qui reste stable aux pH bas (1, 2) et qui a un point de coupure correspondant à un poids moléculaire de 10 000 daltons environ. Après une opération d'échange cationique (22) et d'échange anionique (26), suivie par un traitement au charbon actif (21), l'acide citrique est récupéré et purifié par des cristallisations répétées (40, 42, 50, 53). Pour obtenir un produit de pureté maximale, on peut soutirer (44, 45, 80) une partie de la liqueur mère restante, obtenue après la première étape de cristallisation, et la soumettre à un ou plusieurs traitements additionnels de purification, ou la recycler. La nouvelle souche A. niger CCM 8210 selon l'invention est capable de démarrer la fermentation à un pH très bas, c'est-à-dire inférieur à 4 et par exemple de 2,8 et de continuer la fermentation jusqu'à ce que le pH du substrat soit égal à 1,4 ou moins. A ce moment, le niveau de sucre est inférieur à 1000 mg/l est le rapport acide citrique/sucre résiduel peut atteindre 275 : 1 - 300 : 1. Le milieu de culture obtenu est traité comme décrit.
PCT/SK1996/000014 1995-09-11 1996-09-10 Procede, appareil et souche de micro-organismes pour fabriquer de l'acide citrique WO1997010350A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU70061/96A AU7006196A (en) 1995-09-11 1996-09-10 Process, apparatus and microorganism strain for the manufacture of citric acid

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
SKPV1131-95 1995-09-11
SK113195A SK113195A3 (sk) 1995-09-11 1995-09-11 Spôsob výroby kryštalickej kyseliny citrónovej
SK156-96A SK281100B6 (sk) 1996-02-02 1996-02-02 Mikroorganizmus aspergillus niger ccm 8210
SKPV0156-96 1996-02-02
GBGB9607868.8A GB9607868D0 (en) 1996-04-16 1996-04-16 Process and apparatus for the manufacture of citric acid
GB9607864.7 1996-04-16
GB9607868.8 1996-04-16

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WO1997010350A1 true WO1997010350A1 (fr) 1997-03-20

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19747902A1 (de) * 1997-10-30 1999-05-12 Metallgesellschaft Ag Verfahren zur Herstellung von Citronensäure und/oder Citraten
RU2192460C2 (ru) * 2000-10-20 2002-11-10 Государственное учреждение Всероссийский научно-исследовательский институт пищевых ароматизаторов, кислот и красителей РАСХН Штамм гриба aspergillus niger - продуцент лимонной кислоты
EP0804607B1 (fr) * 1994-03-08 2002-12-18 Cargill, Incorporated Procede de production, de separation et/ou de recuperation d'acide lactique
US6629528B1 (en) 1998-03-17 2003-10-07 Resmed Limited Apparatus for supplying breathable gas
RU2233882C2 (ru) * 2001-11-05 2004-08-10 Государственное учреждение Всероссийский научно-исследовательский институт пищевых ароматизаторов, кислот и красителей РАСХН Способ получения лимонной кислоты
WO2004070022A3 (fr) * 2003-02-05 2004-10-28 Dsm Ip Assets Bv Utilisation de souches d'aspergillus niger deficientes en oxalate pour produire un polypeptide
CN100386439C (zh) * 2005-11-28 2008-05-07 山东柠檬生化有限公司 高纯度柠檬酸的生产方法
CN103952318A (zh) * 2014-03-28 2014-07-30 安徽丰原发酵技术工程研究有限公司 高产柠檬酸黑曲霉菌fy2013及其应用
CN104045551A (zh) * 2014-05-27 2014-09-17 日照金禾博源生化有限公司 一种不合格柠檬酸钠母液的回收方法
JP2018522922A (ja) * 2015-08-10 2018-08-16 パラベル リミテッド 水生生物種からシュウ酸が低減されたタンパク質を抽出するための方法及び系並びにその組成物。
CN110272883A (zh) * 2019-07-03 2019-09-24 上海中溶科技有限公司 一种联产纤维二糖酶和几丁质的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2394031A (en) * 1942-03-14 1946-02-05 Merck & Co Inc Process for the production of citric acid
US3335067A (en) * 1965-05-25 1967-08-08 Miles Lab Process for producing citric acid
US3886041A (en) * 1971-08-18 1975-05-27 Jungbunzlauer Spiritus Production of citric acid by submerged fermentation
DE3502924A1 (de) * 1984-02-03 1985-08-08 Joh. A. Benckiser Gmbh, 6700 Ludwigshafen Verfahren zur gewinnung von citronensaeure

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2394031A (en) * 1942-03-14 1946-02-05 Merck & Co Inc Process for the production of citric acid
US3335067A (en) * 1965-05-25 1967-08-08 Miles Lab Process for producing citric acid
US3886041A (en) * 1971-08-18 1975-05-27 Jungbunzlauer Spiritus Production of citric acid by submerged fermentation
DE3502924A1 (de) * 1984-02-03 1985-08-08 Joh. A. Benckiser Gmbh, 6700 Ludwigshafen Verfahren zur gewinnung von citronensaeure

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0804607B1 (fr) * 1994-03-08 2002-12-18 Cargill, Incorporated Procede de production, de separation et/ou de recuperation d'acide lactique
DE19747902A1 (de) * 1997-10-30 1999-05-12 Metallgesellschaft Ag Verfahren zur Herstellung von Citronensäure und/oder Citraten
DE19747902C2 (de) * 1997-10-30 1999-09-23 Metallgesellschaft Ag Verfahren zur Herstellung von Citronensäure und/oder Citraten
US6087139A (en) * 1997-10-30 2000-07-11 Metallgesellschaft Aktiengesellschaft Process for producing citric acid and/or citrates
US6629528B1 (en) 1998-03-17 2003-10-07 Resmed Limited Apparatus for supplying breathable gas
RU2192460C2 (ru) * 2000-10-20 2002-11-10 Государственное учреждение Всероссийский научно-исследовательский институт пищевых ароматизаторов, кислот и красителей РАСХН Штамм гриба aspergillus niger - продуцент лимонной кислоты
RU2233882C2 (ru) * 2001-11-05 2004-08-10 Государственное учреждение Всероссийский научно-исследовательский институт пищевых ароматизаторов, кислот и красителей РАСХН Способ получения лимонной кислоты
AU2004209618B2 (en) * 2003-02-05 2008-04-17 Dsm Ip Assets B.V. Use of oxalate deficient aspergillus niger strains for producing a polypeptide
WO2004070022A3 (fr) * 2003-02-05 2004-10-28 Dsm Ip Assets Bv Utilisation de souches d'aspergillus niger deficientes en oxalate pour produire un polypeptide
CN100386439C (zh) * 2005-11-28 2008-05-07 山东柠檬生化有限公司 高纯度柠檬酸的生产方法
CN103952318A (zh) * 2014-03-28 2014-07-30 安徽丰原发酵技术工程研究有限公司 高产柠檬酸黑曲霉菌fy2013及其应用
CN103952318B (zh) * 2014-03-28 2017-08-01 安徽丰原发酵技术工程研究有限公司 高产柠檬酸黑曲霉菌fy2013及其应用
CN104045551A (zh) * 2014-05-27 2014-09-17 日照金禾博源生化有限公司 一种不合格柠檬酸钠母液的回收方法
JP2018522922A (ja) * 2015-08-10 2018-08-16 パラベル リミテッド 水生生物種からシュウ酸が低減されたタンパク質を抽出するための方法及び系並びにその組成物。
JP2021138757A (ja) * 2015-08-10 2021-09-16 パラベル ニュートリション インコーポレイテッド 水生生物種からシュウ酸が低減されたタンパク質を抽出するための方法及び系並びにその組成物。
CN110272883A (zh) * 2019-07-03 2019-09-24 上海中溶科技有限公司 一种联产纤维二糖酶和几丁质的方法
CN110272883B (zh) * 2019-07-03 2020-07-21 中溶科技股份有限公司 一种联产纤维二糖酶和几丁质的方法

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