ES2395170B2 - SIMPLIFIED AND OPTIMIZED CULTURE ENVIRONMENT FOR THE PRODUCTION OF ETHANOL AND HYDROGEN, FROM GLYCERINE, BY ESCHERICHIA COLI, TO CONTINUE BIOMASS PRODUCTIVITY - Google Patents

Abstract

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli. # The invention consists of a specific culture medium for the biotransformation process of glycerin in ethanol and hydrogen, using the microorganism E. coli MG1655. This culture medium enhances the growth of the microorganism with the aim of obtaining a concentration of E. coli that allows obtaining higher rates of ethanol production compared to similar processes reported in the literature.

Description

SIMPLIFIED AND OPTIMIZED CULTURE MEDIA FOR THE PRODUCTION OF ETHANOL AND HYDROGEN, FROM GLYCERINE, BY ESCHERICHIA COLL, TO POWER THE



BIOMASS PRODUCTIVITY.

TECHNICAL SECTOR

The present invention falls within the field of biological processes and consists of a culture medium to increase the productivity of biomass of E. coJí with glycerin as the main carbon source.

10 STATE OF THE TECHNIQUE

Glycerin is the main constituent of all fats and oils, it has been the byproduct of greater added value produced during the saponification of oils and fats and transesterification reactions in oleochemical processes and Biodiesel manufacturing, respectively (Kenar 2007).

15 In some European countries, glycerin production has increased significantly due to the increase in Biodiesel production. As a consequence, prices have fallen significantly and most of the companies dedicated to the production of glycerin, due to chemical synthesis, have had to cancel their operations (da Silva et al., 2009), making glycerin overproduction a problem that must be

20 resolved.

The availability of glycerin and its low price have increased interest in its use as a carbon source for different fermentation processes, replacing other carbon sources traditionally used in these processes (Dharmadi et al., 2006). Glycerin is not only abundant and cheap but its high level of reduction, in

25 compared to sugars, it offers the opportunity to obtain reduced chemical compounds such as succinate, ethanol, xylitol, propionate, hydrogen, etc. with higher production yields compared to those obtained from sugars (da Silva et al., 2009).

The biotransformation process allows to transform a compound (raw material)

in another of technical, commercial interest, etc. (product), through enzymes or microorganisms that allow catalyzing the required transformation reactions. The biotransformation process has advantages, compared to traditional chemical synthesis, with respect to its selectivity, specificity and moderate process conditions (pressure, temperature, pH), having repercussions on benefits related to the non-production of unwanted isomers, lower energy expenditure, etc. which finally translates into a more efficient and feasible process from a technical and economic point of view.

Various microorganisms are capable of metabolizing glycerin, as a source of carbon Klebsiella, Citrobacter, Enterobacter, Clostridium, Lactobacillus, Bacillus, Propionibacterium, Anaerobiospirillum, etc., however the commercial use of these organisms is limited by the degree of pathogenicity they possess, nutritional requirements and difficulty for their genetic manipulation (Yazdani and Gonzalez, 2008).

Recently it has been described that E. coli can efficiently ferment glycerin when the pH of the culture medium is acidic, with ethanol and succinate being the main

products of this fermentation, which makes it an attractive model for the use of glycerin in the production of ethanol and hydrogen (Hakobyan et al., 2005; Dharmadi et al., 2006; Murarka et al., 2008; Yazdani et al ., 2008; Gonzalez, 2009; Guebel et al., 2009).

The optimization of the culture medium is an important step in the design of a fermentation process. A desirable result is the reduction in the cost of materials and / or energy.

It is possible to confirm the absence of a culture medium specifically formulated for the biotransformation process of glycerin in ethanol and hydrogen, by means of E. coli under anaerobic conditions. Therefore, the present invention represents an advance in this area since, a specific culture medium was formulated and optimized for the biotransformation process of glycerin in ethanol, using the E. coli MG1655 microorganism. This culture medium enhances the growth of the microorganism with the aim of obtaining a concentration of E. coli that allows obtaining higher rates of ethanol production compared to similar processes reported in the literature. The culture medium developed and optimized in this work can be considered a simple and attractive way to produce the

ethanol and hydrogen using glycerin as a carbon source and Escherichia coli as

biocatalyst, becoming an attractive alternative to future stages of escalation, where low costs and simplicity in the formulation and preparation of a culture medium is important.

References cited:

•

Da Silva, G.P., Mack, M., Conteiro., J. 2009. Glycerol: A promising and abundant carbon source for industrial microbiology. Biotechnology Advances 27: 3 (} - 39.

•

Oharmadi, Y., Murarka, A., Gonzalez, R. 2006. Anaerobic Fermentation of Glycerol by Escherichia coli: A New Platform for Metabolic Engineering. Biotechnology and Bioengineering. 94 (5): 821-829.

•

Gonzalez, R. 2009. Anaerobic Fermentation of Glycerol. United 5tates Patent Application. Pub. No .: U5 2009/0186392 Al.

•

Guebel, O.V., Cánovas, M., Torres, N.V. 2009. Analysis of the Escherichia coli Response to Glycerol Pulse in Continuous, High-Cell Oensity Culture Using a Multivariate Approach. Biotechnology and Bioengineering 102 (3): 91 (} - 922.

•

Hakobyan, M., 5argsyan, H., Bagramyan K. 2005. Proton translocation coupled to formate oxidation in anaerobically grown fermenting Escherichia coli. Biophysical Chemistry 115: 55-61.

•

Kenar, J.A. 2007. Glycerol as a platform chemical: sweet opportunities on the horizon? Lipid Technology 19 (11): 249-253.

•

Murarka, A., Oharmadi, Y., Yazdani, S.S., Gonzalez, R. 2008. Fermentative Utilization of Glycerol by Escherichia coli and Its Implications for the Production of Fuels and Chemicals. Applied and environmental microbiology. 74 (4): 1124-1135.

•

Yazdani, S.S., Gonzalez, R. 2008. Engineering Escherichia coli for the efficient conversion of glycerol to ethanol and co-products. Metabolic Engineering 10: 340

351

DESCRIPTION OF THE INVENTION

The invention consists of a culture medium formulated and optimized to enhance the growth of the biomass responsible for the production of ethanol and hydrogen from glycerin, under anaerobic conditions. The culture medium is composed of glycerin, Na2S04, NaCl, MgS04 · 7H20 and peptone.

Both glycerin and peptone, as well as the inorganic salts that make up the medium are presented in Table 1, where it is possible to observe the appropriate concentration ranges and

Preferred concentration ranges, expressed in g r.

Table 1

Compound

Glycerin

I was born

Peptone

Concentration

Concentration

appropriate (g L-1)

preferred (g L-1)

5-50

8-12

0.02-1.2

0.05-1.05

0-0.03

0, 01-0, 02

0.01-0.05

0, 02-0, 04

2-8

3-6

This medium can be prepared directly or in concentrated solution, preferably 15x, the peptone can be prepared separately.

If necessary, the medium can be supplemented with trace inorganic salts, presented in Table 2, in concentrations that are not inhibitory for E. coli, 15 during this process.

Table 2

Compound Concentration Appropriate concentration (g L-1) preferred (g L-1)

Fe2 (S04) 3

0-0,0045 0-0,00225

MnS04H2O

0-0,0037 0-0.00185

CuS045H2O

0-0,0047 0-0,00235

ZnS047H20

0-0,0053 0-0.00265

(NH4) 6M07024 4H2O

0-0,00055 0-0,000275

CoCI36H2O

0-0,001 0-0,0005

The pH of the culture medium can be adjusted to values between 6.0-6.5; according to the requirements of the process that you want to carry out. Table 3 shows a comparison of the fermentation parameters S obtained in the proposed medium, with respect to Murarka et al. (2008) Table 3

Parameter Units In this work Murarka et al. (2008) Strain

0.0313iO.0015 0.040iO.003 () MG1655

Glycerol% 84.46 ± 0.023 = 80 + MG1655 consumed

-

one

getane! gglycerol 0.581 0.461 MG1655

yx / s gcel glycerol -1 0.059 ± 0.019 0.0329 ± 0.0029 MG1655

-lh-l

getanol 0.128 = 0.146 + MG1655

gcel r1d-1 0.165iO.0126 = 0.0752 + MG1655

+ calculated from Murarka et al., 2008.

The proposed medium allows ethanol and hydrogen to be produced in a simple and attractive way, reducing both the cost of formulation and the preparation time in larger-scale uses.

As seen in Figure 2, the stationary phase for biomass growth was reached in 70 hours of fermentation compared to the 97 hours needed in the non-optimized medium (Figure 1). On the other hand, the production of maximum ethanol was reached at 80 hours for the optimized medium (Figure 2) versus the 120 hours needed for the non-optimized medium (Figure 1). The fermentation parameters were calculated for the experiments performed with the optimized culture medium. As a control, these results were compared with previously disclosed data, as shown in Table 1. The culture medium used by Murarka was a modified minimum medium designed by Neidhardt et al. (1973).

The results obtained with the optimized culture medium are quite significant compared to those reported by Murarka et al. (2008) since Ox, Yx / s and Yp / s values are reached are 2.19, 1.79 and 1.26 times higher, respectively, than the data disclosed by these authors for a similar process.

Regarding hydrogen production, they are in the order of magnitude compared to those reported in literature, for similar processes.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1.- Shows the fermentation kinetics obtained for non-optimized culture medium: biomass growth (o) (it appears multiplied five times), glycerin consumption (+) and ethanol production (.).

Figure 2.- Shows the fermentation kinetics obtained for optimized culture medium: biomass growth (o) (it appears multiplied five times), glycerin consumption (+) and ethanol production (.).

Claims (30)

one.

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, under anaerobic conditions to enhance the productivity of biomass, composed of glycerin, Na2S04, NaCI, MgS04 · 7H20 and peptone.

2.

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 1, wherein the glycerin concentration is in a range between 5 and 50 g L-1 •

3.

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 1, wherein the concentration of Na2S04 is in a range between 0.02 and 1.2 g L -one.

Four.

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 1, wherein the concentration of NaCI is in a range between 0 and 0.03 g L-1 •

5.

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 1, wherein the

MgS04 · 7H20 concentration is in a range between 0.01 and 0.05 g r.

6.

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 1, wherein the concentration of Peptone is in a range between 2 and 8 g L-1.

7.

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claims 1 and 2, wherein the preferred glycerin concentration is in a range of between 8 and 12 g L- one•

8.

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claims 1 and 3, wherein the preferred concentration of Na2S04 is in a range between 0.05 and 1, 05 g L-1 •

9.

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claims 1 and 4, wherein the preferred NaCl concentration is in a range between 0.01 and 0, 02 g r1 •

10.

Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claims 1 and 5, wherein the concentration of MgS04 · 7H20 preferred is in a range of between 0.02 and 0.04

L "l

eleven.

Simplified and optimized culture medium for the production of ethanol and hydrogen, to

starting from glycerin, by Escherichia coli, according to claims 1 and 6, wherein the 1 Preferred Peptone concentration is in a range between 3 and 6 g r. 12. Simplified and optimized culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia colí, according to claims 1 to 11, prepared 10 directly or in concentrated solution, preferably 15x, the peptone can be prepared separately. 13. Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claims 1 to 12, supplemented with inorganic salts 14. Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 13, wherein the concentration of Fe2 (S04h is found in a range between 0 and 0.0045 g r. 15. Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 13, wherein the concentration of MnS04 H20 is 20 is in a range between 0 and 0.0037 g r.

16.

Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 13, wherein the concentration of CUS04 5H20 is in a range between 0 and 0.0047 g r1 •

17.

Culture medium for the production of ethanol and hydrogen, from glycerin, by

Escherichia coli, according to claim 13, wherein the concentration of ZnS04 7H20 is in a range between 0 and 0.0053 g r1 •

18.

Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 13, wherein the concentration of (NH4) 6Mo7024 4H20 is in a range between O and 0.0055 g L " l.

19.

Culture medium for the production of ethanol and hydrogen, from glycerin, by

Escherichia coli, according to claim 13, wherein the concentration of CoCI3 6H20 is in a range between 0 and 0.001 g r1.

twenty.

Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia colí, according to claim 14, wherein the concentration of Fe2 (S04h preferred is in a range between 0 and 0.00225 g r1.

twenty-one.

Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 15, wherein the concentration of MnS04 H20 preferred is in a range between 0 and 0.00185 g r1.

22

Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coJi, according to claim 16, wherein the preferred concentration of CUS04 5H20 is in a range between 0 and 0.00235 g L-1.

2. 3.

Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia coli, according to claim 17, wherein the concentration of ZnS04 7H20 preferred is in a range between 0 and 0.00265 g L-1 •

24.

Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia colí, according to claim 18, wherein the concentration of (NH4) 6M07024 4H20 is in a range between 0 and 0.00275 g r1 .

25.

Culture medium for the production of ethanol and hydrogen, from glycerin, by Escherichia colí, according to claim 19, wherein the preferred concentration of CoCI3 6H20 is in a range between 0 and 0.0005 g r1 •

26.

Use of the simplified and optimized culture medium, according to claim 1, in processes of ethanol and hydrogen production from glycerin by Escherichia colí.

27.

Use of the simplified and optimized culture medium, according to claims 2 to 6, in ethanol and hydrogen production processes from glycerin by Escherichia coli.

28.

Use of the simplified and optimized culture medium, according to claims 7 to 12, in ethanol and hydrogen production processes from glycerin by Escherichia coli.

29.

Use of the simplified and optimized culture medium, according to claims 13, in ethanol and hydrogen production processes from glycerin by Escherichia colí.

30

Use of the simplified and optimized culture medium, according to claims 14 to 19, in ethanol and hydrogen production processes from glycerin by Eseheriehia eoli.

31.

Use of the simplified and optimized culture medium, according to claims 20 to 25, in ethanol and hydrogen production processes from glycerin by Eseheriehia eoli.