Method For Producing Citric Acid by Degrading Roughages with the Rumen Fungus-methanogen Co-culture from Qinghai Yaks

Abstract

The present disclosure relates to the technical field of biotechnology renewable energy, and more specifically, to a method for producing citric acid by degrading roughage with natural symbiotic mixed culture. The mixed culture YakQH5 is composed of anaerobic fungi (Neocallimastix frontalis) and methanogens (Methanobrevibacter gottschalkii). It was collected in the China General Microbiological Culture Collection Center on Mar. 9, 2020, with the collection number of CGMCC No. 19299. The mixed culture YakQH5 can degrade 15 kinds of roughage respectively and produce a large amount of citric acid. Especially when alfalfa is used as substrate, the yield of citric acid is as high as 46.0 mm. Adding compound antibiotics in the fermentation process can also prevent the mixed culture from being polluted by bacteria in the fermentation process and further improve the efficiency of anaerobic fermentation. The mixed culture YakQH5 has important industrial application value.

Claims

1. A method for producing citric acid by degrading roughage with the natural fungus-methanogen co-cultures from the rumen of Qinghai yaksthe N. frontalis+M. gottschalkii co-culture YakQH5. The N. frontalis+M. gottschalkii co-culture YakQH5 is composed of anaerobic fungus and methanogen, and the method comprises: S1. Preparation of the N. frontalis+M. gottschalkii co-culture YakQH5 inoculum: inoculating the N. frontalis+M. gottschalkii co-culture YakQH5 into an anaerobic medium with wheat straw as substrate at an inoculation amount of 10% v/v, adding compound antibiotics and performing anaerobic culture to obtain high-activity inoculum; and S2. Fermentation and degradation of roughage to produce citric acid: absorbing the inoculum prepared in step S1, inoculating it into an anaerobic medium with 1% w/v roughage as substrate according to an inoculation amount of 10% v/v, adding 1% v/v compound antibiotics at the same time, and culturing; the mixed culture YakQH5 is collected in China General Microbiological Culture Collection Center on Mar. 9, 2020, the collection number is CGMCC No. 19299 and the collection address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 yard, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355; in the step S1, a temperature of anaerobic culture is 39 C., and a time is 72 hours; in step S2, a temperature of anaerobic culture is 39 C., and a time is 5 days; the compound antibiotics are penicillin sodium and streptomycin sulfate, and the concentrations in the anaerobic medium are 1600 IU/mL and 2000 IU/mL respectively; a formula of the anaerobic medium is as follows: yeast extract 1.0 g, peptone 1.0 g, NaHCO.sub.3 7.0 g, resazurin (1.0 g/1) 1 ml, L-cysteine hydrochloride 1.7 g, rumen fluid collected before morning feeding 8000g, 170 mL of supernatant after centrifugation at 4 C. for 20 min, salt solution I 82.5 mL, salt solution II 16.5 mL, and distilled water to make up to 1000 mL; a formula of the salt solution I is as follows: NaCl 6 g, (NH.sub.4).sub.2SO.sub.4 3 g, KH.sub.2PO.sub.4 3 g, CaCl.sub.2.Math.2H.sub.2O 4 g, MgSO.sub.4.Math.2H.sub.2O 0.6 g, and distilled water to make up to 1000 mL; and a formula of the salt solution II is as follows: K.sub.2HPO.sub.4 4 g, and distilled water to make up to 1000 mL; the step S2 comprises deoxygenating after adding various substrates respectively, filling carbon dioxide, and sterilizing at high temperature and high pressure; the substrates added in step S2 are one or more of wheat straw, flax straw, potato straw, cotton straw, soybean straw, sainfoin straw, reed straw, sisal, bran, wheat husk, filter paper, tomato residue and alfalfa.

2. The method of claim 1, wherein the substrate added in step S2 is alfalfa.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The experimental methods used in the following embodiments are conventional methods unless otherwise specified.

[0022] The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0023] The culture media used in the following examples are as follows.

[0024] The formula of the anaerobic medium was as follows: yeast extract 1.0 g, peptone 1.0 g, NaHCO.sub.3 7.0 g, resazurin (1.0 g/l) 1 mL, L-cysteine hydrochloride 1.7 g, rumen fluid collected before morning feeding 8000g, 170 mL of supernatant after centrifugation at 4 C. for 20 min, salt solution I 165 mL, salt solution II 165 mL, and distilled water to make up to 1000 mL.

[0025] The formula of the salt solution I was as follows: NaCl 6 g, (NH.sub.4).sub.2SO.sub.4 3 g, KH.sub.2PO.sub.4 3 g, CaCl.sub.2.Math.2H.sub.2O 0.4 g, MgSO.sub.4.Math.2H.sub.2O 0.6 g, and distilled water to make up to 1000 mL.

[0026] The formula of the salt solution II was follows: K.sub.2HPO.sub.4 4 g, and distilled water to make up to 1000 mL.

[0027] Subculture medium: 1% w/v crushed and air-dried wheat straw was added to the anaerobic medium. Then the sterilization was performed after deaeration.

[0028] Deaeration method: the anaerobic pipe or anaerobic bottle was connected to the air extraction device with vacuum pump and high-purity CO.sub.2 through the needle to deaerate the culture medium. Firstly, when the gas in the vacuum pump extraction pipe reached negative pressure, the color of the culture medium changed, and then the high-purity CO.sub.2 was filled. Each pipe was pumped and inflated for 3 times, of which the first time was about 15 min, and the other two times were 5 min each time. After the last inflation, the sterile needle was used to deflate again to balance the internal and external pressure of the anaerobic pipe, and the sterilization was performed at 121 C. high temperature, high pressure and damp heat for 20 minutes for standby.

[0029] Anaerobic fermentation tank, also known as anaerobic fermentation bottle, is a closed tank, in which the process of material liquid fermentation and biogas production is completed. It is mainly used to meet the living conditions of microorganisms and make them live in a suitable environment, so as to achieve the purpose of vigorous fermentation and high gas production.

Embodiment 1: Preparation of the N. frontalis+M. Gottschalkii Co-Culture YakQH5

[0030] 1 mL the N. frontalis+M. gottschalkii co-culture YakQH5 was taken and inoculated into 9 ml of anaerobic medium with air dried and crushed wheat straw as substrate in Heinz anaerobic pipe. At the same time, 0.1 mL of compound antibiotics (1600 IU/mL penicillin and 2000 IU/ml streptomycin sulfate) were added. Anaerobic culture was performed at 39 C. for 72 h to reach the growth peak. At this time, the fermentation broth was a highly active inoculum.

Embodiment 2: Fermentation of the N. frontalis+M. Gottschalkii Co-Culture YakQH5 to Produce Citric Acid

[0031] 45 mL liquid basic medium was filled in a 100 mL volume anaerobic fermentation bottle. A total of 15 kinds of roughage, including 0.5 g crushed air-dried wheat straw, flax straw, potato straw, cotton straw, soybean straw, sainfoin straw, reed straw, sisal, bran, melon seed husk, peanut husk, wheat husk, filter paper, tomato residue and alfalfa, was used as substrates respectively. Then the deaeration and sterilization were performed. The mixed culture YakQH5 subcultured for 72 h was inoculated into the above-mentioned anaerobic medium with each roughage by drawing 5 mL with a sterile syringe. At the same time, 0.5 mL of compound antibiotics (1600 IU/mL penicillin and 2000 IU/mL streptomycin sulfate) were added. Anaerobic culture was performed at 39 C. for 5 days. Three parallel experiments were set up to measure the citric acid concentration in the anaerobic bottle every 24 hours. Citric acid was determined by liquid chromatography.

[0032] The liquid chromatograph (LC2030 PLUS, Shimadzu, Japan) was used and equipped with Acclaim 120 C18 3 um column (21 mm50 mm). The detection wavelength was 210 nm, column temperature was 25 C., UV detector temperature was 40 C., and mobile phase A: potassium phosphate aqueous solution (pH 2), mobile phase B: acetonitrile, flow rate 0.5 m/min, 0 min 99% A-1% CB, 20 m 80% A-20% B, injection volume 10 L. The experimental results showed that the N. frontalis+M. gottschalkii co-culture YakQH5 degraded 15 kinds of roughages and produced high concentration citric acid. The yield of citric acid produced with alfalfa as substrate was significantly higher than that of other substrates. The specific results are as follows.

TABLE-US-00001 TABLE 1 Yield of citric acid produced by degradation of 15 kinds of roughages with the N. frontalis + M. gottschalkii co-culture YakQH5 from Qinghai yaks. Citric acid concentration (mM) No. Roughage substrate 2d 3d 4d 5d Average 1 Wheat straw 5.0.sup.c 11.7.sup.b 23.6.sup.c 32.8.sup.d 18.3.sup.b 2 Flax straw 3.9.sup.b 10.9.sup.b 21.5.sup.c 28.2.sup.c 16.1.sup.b 3 Potato straw 3.0.sup.b 12.5.sup.b 26.1.sup.c 31.0.sup.d 18.2.sup.b 4 Cotton straw 1.0 8.2.sup.b 15.9.sup.b 20.0.sup.c 11.3.sup.b 5 Soybean straw 3.8.sup.b 9.5.sup.b 16.4.sup.b 27.1.sup.c 14.2.sup.b 6 Sainfoin straw 9.1.sup.d 20.3.sup.d 38.6.sup.d 40.1.sup.d 27.0.sup.c 7 Reed straw 2.0.sup.b 14.1.sup.c 20.5.sup.c 24.6.sup.c 15.3.sup.b 8 Sisal 3.5.sup.b 13.8.sup.b 22.0.sup.c 27.8.sup.c 16.8.sup.b 9 Bran 2.0.sup.b 7.6 10.5 16.3.sup.b 9.1 10 Melon seed husk 11 Peanut husk 12 Wheat husk 4.6.sup.c 12.0.sup.c 18.7.sup.b 20.7.sup.c 14.0.sup.b 13 Filter paper 3.2.sup.b 10.2.sup.b 17.0.sup.b 21.4.sup.c 12.9.sup.b 14 Tomato residue 1.0 5.1 9.3 11.5 6.7 15 Alfalfa 12.3.sup.d 26.5.sup.d 37.9.sup.d 46.0.sup.d 30.7.sup.d Note: , means not measured. .sup.a, .sup.b, .sup.c and .sup.d indicate statistical difference (p < 0.05).

[0033] The experimental results are shown in Table 1. The N. frontalis+M. gottschalkii co-culture YakQH5 degraded 15 kinds of roughage respectively during the 5-day culture period to produce citric acid, and the highest yields are as follows. The yield of citric acid produced by fermentation with wheat straw as substrate reached 32.8 mM, the yield of citric acid produced by fermentation with flax straw as substrate reached 28.2 mM, the yield of citric acid produced by fermentation with potato straw as substrate reached 31.0 mM, the yield of citric acid produced by fermentation with cotton straw as substrate reached 20.0 mM, the yield of citric acid produced by fermentation with soybean straw as substrate reached 27.1 mM, the yield of citric acid produced by fermentation with sainfoin straw as substrate reached 40.1 mM, the yield of citric acid produced by fermentation with reed straw as substrate reached 24.6 mM, the yield of citric acid produced by fermentation with sisal as substrate reached 27.8 mM, the yield of citric acid produced by fermentation with bran as substrate reached 16.3 mM, the yield of citric acid produced by fermentation with wheat husk as substrate reached 20.7 mM, the yield of citric acid produced by fermentation with filter paper as substrate reached 21.4 mM, the yield of citric acid produced by fermentation with tomato residue as substrate reached 11.5 mM, and the yield of citric acid produced by fermentation with alfalfa as substrate reached 46.0 mM.

[0034] It can be seen from the above embodiments that the natural fungusmethanogen co-culturesthe N. frontalis+M. gottschalkii co-culture YakQH5 from the rumen of Qinghai yaks can degrade 15 kinds of roughage and produce a large amount of citric acid at the same time. In particular, the citric acid produced by degraded alfalfa reaches the highest value of 46.0 mm, which has important industrial application value.