Method for the production of carboxylic acids under unsterile conditions

Abstract

The aim of the invention is to provide a method for biotechnological production of carboxylic acids, in which the acid-forming micro-organisms are cultured in an unsterile manner in a submerged phase containing waste water containing all carbon and nutrient medium components necessary for the production of the carboxylic acid, which method avoids the disadvantages of known methods and enables high product concentrations and productivity while at the same time the resources of water and power are being conserved. This aim is achieved, according to the invention, in that micro-organisms are used that are cultured under unsterile conditions in a culture medium containing waste water with the addition of carbon-rich compounds.

Claims

1. A method for biotechnological production of carboxylic acids, wherein the method comprises: culturing of microorganisms under unsterile conditions in a culture medium containing waste water, wherein the waste water comprises carbon-rich compounds and/or the method comprises adding carbon-rich compounds to the waste water, the microorganisms comprise strains of the yeast type Yarrowia lipolytica, the carboxylic acids to be produced comprise at least one substance selected from the group consisting of aconitic, malic, succinic, citric, fumaric, isocitric, α-ketoglutaric, oxaloacetic, pyruvic, and itaconic acids, and the carbon-rich compounds comprise at least one substance selected from the group consisting of monoglycerides, diglycerides, triglycerides, fatty acids, fatty acid methyl or ethyl esters, ethanol, methanol, glycerol, glucose, fructose, galactose, lactose, lactic acid, saccharose, xylose, arabinose, and n-alkanes, wherein the unsterile conditions comprise that before, during and after cultivation, no chemical or thermal sterilization measures and pasteurization measures are applied; and wherein the method comprises at least one pre-cultivation step for producing inoculum of the yeast Yarrowia lipolytica in an unsterile manner.

2. The method according to claim 1, wherein the microorganisms comprise the yeast strain Yarrowia lipolytica H181 (DSM 7806).

3. The method according to claim 1, wherein the waste water is a water that is contaminated through use and consumption.

4. The method according to claim 1, wherein the waste water is fed to a reaction vessel in a volume proportion of 5-100%.

5. The method according to claim 1, wherein the culturing of microorganisms is conducted in an unsterile manner and no measures and devices are provided for production and maintenance of a germ-free and sterile state and for prevention of penetration of external germs into the culture medium.

6. The method according to claim 1, wherein the culturing of microorganisms is conducted in a reaction facility comprising reaction vessels that are selected from the group consisting of stirrer reactors, airlift reactors, bubble columns, aeration tanks and sequencing batch reactors.

7. The method according to claim 1, wherein the culturing of microorganisms is conducted at a pH of 2 to 8.

8. The method according to claim 1, wherein the culturing of microorganisms is conducted at a temperature of 5 to 45° C.

9. The method according to claim 1, wherein the method comprises a step of neutralizing with an agent selected from the group consisting of CaCO.sub.3, Ca(OH).sub.2, KOH, NaOH, Na.sub.2CO.sub.3, and NaHCO.sub.3.

10. The method according to claim 1, wherein a dissolved oxygen concentration is set at a range of pO2≤1% to 100% during the culturing of microorganisms.

11. The method according to claim 1, wherein the culturing of microorganisms is conducted as a batch, fedbatch, semi-continuous repeated fedbatch or continuous process.

12. The method according to claim 1, wherein the culturing of microorganisms takes place without prior immobilisation of the microorganisms.

13. The method according to claim 1, wherein the waste water is selected from the group consisting of grey water, industrial wastewater, municipal waste water, waste water from cleaning stages of a kitchen and catering operation, discharges from commercial oil/fat separators and discharges from a pre-clarification stage of waste water treatment plants for communal waste waters.

14. A method for biotechnological production of carboxylic acids, wherein the method comprises: culturing of microorganisms under unsterile conditions in a culture medium containing waste water, wherein the waste water comprises carbon-rich compounds and/or the method comprises adding carbon-rich compounds to the waste water, wherein the unsterile conditions comprise that before, during and after cultivation, no chemical or thermal sterilization measures and pasteurization measures are applied, the microorganisms comprise the yeast strain Yarrowia lipolytica H181 (DSM 7806), the carboxylic acids to be produced comprise at least one substance selected from the group consisting of aconitic, malic, succinic, citric, fumaric, isocitric, α-ketoglutaric, oxaloacetic, pyruvic and itaconic acids, and the carbon-rich compounds comprise at least one substance selected from the group consisting of monoglycerides, diglycerides, triglycerides, fatty acids, fatty acid methyl or ethyl esters, ethanol, methanol, glycerol, glucose, fructose, galactose, lactose, lactic acid, saccharose, xylose, arabinose, and n-alkanes; and wherein the method comprises at least one pre-cultivation step for producing inoculum of the yeast Yarrowia lipolytica H181 (DSM 7806) in an unsterile manner.

15. The method according to claim 14, wherein the waste water is a water that is contaminated through use and consumption.

16. The method according to claim 14, wherein the waste water is selected from the group consisting of grey water, industrial wastewater, municipal waste water, waste water from cleaning stages of a kitchen and catering operation, discharges from commercial oil/fat separators and discharges from a pre-clarification stage of waste water treatment plants for communal waste waters.

17. The method according to claim 14, wherein the waste water is fed to a reaction vessel in a volume proportion of 5-100%.

18. The method according to claim 14, wherein the culturing of microorganisms is conducted in an unsterile manner and no measures and devices are provided for production and maintenance of a germ-free and sterile state and for prevention of penetration of external germs into the culture medium.

Description

EXAMPLE 1

(1) Obtaining the Pre-Culture

(2) The strain Yarrowia lipolytica H181 (DSM 7806) was cultured on a known nutrient medium suitable for yeasts (e.g. peptone yeast extract agar or reader agar) for 24 to 48 h at 30° C.

(3) From the grown agar culture, 2 to 3 inoculation loops were seeded on 100 ml of a sterile pre-culture solution produced with distilled water with the following composition:

(4) TABLE-US-00001 NH.sub.4Cl 3.00 g/l KH.sub.2PO.sub.4 0.70 g/l MgSO.sub.4 × 7H2O 0.35 g/l NaCl 0.10 g/l CaCl.sub.2 × 2H2O 0.13 g/l FeSO.sub.4 × 7H2O  3.5 mg/l Thiamine × HCl   1 mg/l Trace salt solution   5 ml/l Sunflower oil   50 g/l

(5) The trace salt solution of the pre-culture medium had the following composition:

(6) TABLE-US-00002 CuSO.sub.4 × 5H2O 4.0 g/l MnSO.sub.4 × 5H2O 4.0 g/l ZnCl.sub.2 2.1 g/l CoSO.sub.4 × 7H2O 0.5 g/l H.sub.3B0.sub.3 5.7 g/l

(7) The pre-culture was cultured and at ph 5-6 in 500 ml Erlenmeyer flask with baffles at 30° C. for 48 h on a shaking apparatus with a shaking frequency of 120 to 140 rpm.

(8) Implementation of Main/Production Culture

(9) From the pre-culture, the seeding of 30 ml of inoculum with an inoculation ratio of 1:10 in a 0.5 l stirrer reactor with 300 ml of working volume was conducted for the implementation of the unsterile, submerged main/production cultivation, wherein the terms main and production culture are used as synonyms. The production medium contained the following components:

(10) TABLE-US-00003 NH.sub.4Cl 3.00 g/l KH.sub.2PO.sub.4 0.70 g/l MgSO.sub.4 × 7H2O 0.35 g/l CaCl.sub.2 × 2H2O 0.13 g/l FeSO4 × 7H2O  3.5 mg/l Thiamine × HCl   1 mg/l Trace salt solution   1 ml/l

(11) (Composition of the Trace Salt Solution as for the Pre-Culture)

(12) As a carbon source, the production medium contained an old deep-frying oil, which was previously used as deep-frying oil for two days for the production of potato chips. The old deep-frying oil had the following fatty acid pattern (mass percentages):

(13) TABLE-US-00004 Myristic acid (14:0) 5.4% Palmitic acid (16:0) 0.2% Stearic acid (18:0) 1.6% Oleic acid (18:1; w9c) 58.7% Vaccenic acid (18:1; w7c) 3.5% Linoleic acid (18:2) 19.6% Linolenic acid (18:3) 8.7% Arachidonic acid (20:0) 0.4% Eicosenoic acid (20:1; w9c) 0.9% Other fatty acids 1.0%

(14) The waste water from the discharge of an oil and fat separator of a commercial canteen and catering operation was used as the liquid phase in order to produce the production medium. The discharge of the oil/fat separator was characterized by the following waste water-related parameters:

(15) TABLE-US-00005 Parameter Ph   6.3 DOC   6.7 mg O.sub.2/l Conductivity  2.17 ms/cm COD 2830 mg/l BOD.sub.5 1611 mg/l TOC  954 mg/l TSS  576 mg/l TN  32 mg/l

(16) All nutrient medium components of the production medium were dissolved in the waste water and added in an unsterile manner to the 0.5 l stirrer reactor. The initial concentration of the old deep-frying oil in the stirrer reactor was 40 g/l and was added in an unsterile manner in fedbatch operation in stages of 20 g/l up to a final concentration of 140 g/l. The production cultures were implemented as a duplicate batch at 30° C., stirrer speed=900 rpm and a dissolved oxygen concentration of pO.sub.2=50%. The dissolved oxygen concentration was regulated via a gas mixture of air/nitrogen/oxygen, wherein the ventilation of the stirrer reactors was conducted in an unsterile manner without feed and exhaust air filters, with a total gas flow of 0.5 I/min. The ph value was established at ph 5 with 20% NaOH.

(17) In comparison, sterile double batches with a) distilled water (including all nutrient components, sterilised at 121° C. for 20 mins) instead of waste water and old deep-frying oil (pasteurised at 80° C. for 20 mins), and b) oil/fat separator discharge (including all nutrient components, sterilised at 121° C. for 20 mins) and old deep-frying oil (pasteurised at 80° C. for 20 mins) were realised.

(18) When the nitrogen source, ammonium sulphate, was used up after 21 h, the accumulation of citric acid and isocitric acid began in the culture solutions. After 166 h, the cultivations were ended. For the unsterile batch with waste water (discharge from oil/fat separator), a carboxylic acid formation of 137 g/l citric acid and 11 g/l of the by-product isocitric acid were determined (average values from two cultivations). This corresponded to a productivity of 0.83 g/l*h. When waste water is used under sterile conditions, the product quantities of carboxylic acids were only insignificantly increased; with distilled water, the lowest concentrations of citrate were formed (Table 1).

(19) TABLE-US-00006 TABLE 1 The production of citric acid of yarrowia lipolytica H181 (DSM 7806) under unsterile culture conditions (average value from two identical test batches respectively) Example 2 Culture Isocitric acid Productivity Selectivity Batch duration Citric acid (g/l) (g/l) citrate (g/l * h) citrate (%) unsterile Discharge oil 166 h 137 ± 8.5 11.2 ± 1.4 0.83 ± 0.05 92.4 ± 0.5 separator + old deep- frying oil sterile a.) Distilled 166 h 125 ± 4.6 15.2 ± 3.7 0.75 ± 0.03 89.3 ± 2.0 water + old deep-frying oil b.) Discharge 166 h 145 ± 9.6 14.9 ± 0.6 0.88 ± 0.06 90.7 ± 0.2 oil separator + old deep- frying oil

(20) Cultivation took place as described in example 1, wherein a waste water (discharge of oil/fat separator) was used in the main culture, which had the following composition:

(21) TABLE-US-00007 Parameter PH   5.9 DOC   1.7 mg O.sub.2/l Conductivity   3.0 ms/cm COD 1760 mg/l BOD.sub.5 1036 mg/l TOC  676 mg/l TSS  315 mg/l TN  29 mg/l

(22) The main culture was achieved as a semi-continuous cultivation (repeated feedback operation) in a 15 l stirrer reactor. Here, cycle 1 was started with 9 l of working volume, which contained 900 ml of the pre-culture of Y. lipolytica H181 (DSM 7806) and an initial old deep-frying oil concentration of 40 g/l. The carbon source was fed up to a summary final concentration of 120 g/l old oil.

(23) In contrast to example 1, the ph value 5 was established with 45% NaOH, and the oxygen supply of the yeasts was realised by means of a ventilation quantity of 4 l/min with a stirrer speed of 800 rpm. The dissolved oxygen concentration for the entire cultivation was pO.sub.2≤20%.

(24) When the nitrogen source, ammonium sulphate, was used up after 20 h, the accumulation of citric acid and isocitric acid could be registered. After a culture duration of 70 h, cycle 1 of the main cultivation was ended. The volume of the culture solution was 10.5 l and contained 77 g/l of citric acid and 3.8 g/l isocitric acid. This corresponded to a productivity of 1.1 g/l*h and selectivity of 95.2% for citric acid.

(25) Following the completion of cycle 1, 9.5 l of the culture solution were drained down to a residual volume of 1 l, and were filled in an unsterile manner with 8 l of the oil/fat separator discharge, including the nutrient medium components of the production medium. The yeast biomass of Y. lipolytica H181 contained in the residual volume of cycle 1 acted as an inoculum for the continuation of the cultivation in the second cultivation cycle.

(26) In cycle 2, 150 g/l of old deep-frying oil was added to the stirrer reactor in fedbatch mode as an external source of carbon. The conditions of cycle 1 were maintained with regard to the ph value and the oxygen supply. When the nitrogen source, ammonium sulphate, was repeatedly used up after 20 h in cycle 2, the formation of citric acid and isocitric acid by the yeast Yarrowia lipolytica H181 began. When cycle 2 was halted after a cycle duration of 95 h, with a total cultivation time of 165 h over both cycles, the concentration of citric acid was 91 g/l, and of isocitric acid, 4.4 g/l in a 10.6 l culture solution. Thus, the productivity (0.96 g/l*h) and selectivity (95.4%) of the citrate formation in cycle 2 were almost identical to the results of cycle 1.

(27) Overall, in both unsterile cycles, 20.1 l of culture solution could be harvested, which contained 1.7 kg of citric acid.

EXAMPLE 3

(28) The cultivation described in example 2 was modified such that the pre-culture was also obtained with the use of a waste water (oil/fat separator discharge). In addition, in contrast to example 2, the unsterile main culture was achieved with untreated waste water from a commercial canteen and catering operation, which was introduced as an infeed into an oil/fat separator. The waste water used (oil/fat separator infeed) had the following composition:

(29) TABLE-US-00008 Parameter ph   5.8 DOC   0.6 mg O.sub.2/l Conductivity   2.9 ms/cm COD 5240 mg/l BOD.sub.5 1661 mg/l TOC 1095 mg/l TSS  191 mg/l TN  117 mg/l

(30) The main culture was conducted as fedbatch cultivation in a 15 l stirrer reactor with a 9 l initial volume. The initial old deep-frying oil concentration was 35 g/l and was increased through the continuous addition of substrate up to 175 g/l after a culture duration of 120 h. In contrast to example 2, the stirrer speed was reduced to 600 rpm. The dissolved oxygen concentration was pO.sub.2>5% during cultivation.

(31) After 21 h, the ammonium nitrogen source was used up; from this point onwards, the citrate and isocitrate began to accumulate. When the main cultivation was halted after 166 h, 181 g/l citric acid and 5.4 g/l isocitric acid could be measured in the culture broth. This corresponded to a productivity of 1.1 g/(l*h) for citric acid. The selectivity for citrate was 97%.

EXAMPLE 4

(32) The cultivation was repeated, as described in example 2, wherein a municipal waste water (septic tank discharge) was used as the liquid phase in the main culture. The municipal waste water had the following composition:

(33) TABLE-US-00009 Parameter Ph 7.2 DOC 0.76 mg O.sub.2/l Conductivity 1.66 ms/cm COD  486 mg/l BOD.sub.5  287 mg/l TOC  163 mg/l TSS   65 mg/l TN   91 mg/l E. coli 4.6 × 106 MPN/100 ml

(34) In contrast to example 2, the unsterile main culture was conducted as fedbatch cultivation (15 l stirrer reactor with 9 l working volume). Here, old deep-frying oil was added as a carbon source up to a final concentration of 180 g/l, starting from an initial substrate concentration of 40 g/l. The cultivation was conducted at ph 5, which was set with 45% NaOH and with a stirrer speed of 600 rpm. The air gasification rate was 4 I/min. Thus, the dissolved oxygen concentrations of pO.sub.2>3% could be maintained over the entire culture duration.

(35) After the NH4-N source was used up at 21 h, during the following cultivation of 56 h, 95 g/l citric acid and 2 g/l isocitrate were formed from Y. lipolytica H181 (DSM 7806), corresponding to a citric acid formation rate of 1.2 g/(l*h). The continuation of the cultivation up to 189 h led to a further increase in the citric acid concentration up to 133.7 g/l. With isocitric acid measured simultaneously at 4.8 g/l, a selectivity of 96.5% could be determined for the citrate formation. The productivity for citrate was 0.71 g/(l*h) for the entire period.

(36) At the end of cultivation, no further evidence was found of E. coli germs (sum parameter of pathogenic bacteria). This is an indication of the hygienic impact of the yeast-based citrate production on waste waters.

(37) In examples 1 to 4 above, the following abbreviations are used:

(38) Ph ph value

(39) DOC Dissolved oxygen content

(40) COD Chemical oxygen demand

(41) BODs Biological oxygen demand (after 5 days)

(42) TOC Total organic carbon

(43) TSS Total suspended solids

(44) TN Total nitrogen

(45) E. coli Escherichia coli