ES2587449B2 - Procedure for the production of bioethanol from chitosan through the use of the nematophagous fungus Pochonia chlamydosporia - Google Patents

Abstract

The present invention relates to a process for the production of ethanol from a chitosan source comprising the use of Pochonia chlamydosporia and the use of said fungus for the degradation of shellfish residues, also obtaining fungal biomass for agrobiotechnological use.

Description

PROCEDURE FOR THE PRODUCTION OF BIOETHANOL FROM CHITOSANE THROUGH THE USE OF POCHONIA CHLAMYOOSPORIA NEMATOPHAGUS HONGO.

FIELD OF THE INVENTION

The present invention falls within the field of biotechnology in general, and in particular, relates to a process for the production of bioethanol from chitosan by the use of Pochonia chlamydosporia.

STATE OF THE TECHNIQUE

The growing search for biofuel production techniques is a strategic field for many countries, due to the problems generated by dependence on petroleum-derived fuels. Although oil is an energy source with intrinsic qualities such as its extraction, good transportability, versatility and low cost, it is a product obtained from the transformation of biomass over 200 million years and its quantity is finite. The decrease in oil reserves and the increase in their price since 2005 explain the new policies that have generated a continuous sustained increase in the demand for biofuels as a substitute (Wright, B. (2014). Global Biofuels: Key to the Puzzle of Grain Market Behavior, Journal of Economic Perspectives, 28 (1), 73-98. Doi: 1 O. t 257 / jep.28.1. 73).

The term biofuel refers to liquid or gaseous fuels, which are produced from biomass. In 2006, liquid biofuels accounted for approximately 1% of global renewable energy. However, this scenario has been transformed very quickly in most of the major energy consuming countries, which are adopting policies that favor greater use of biofuels in the next decade. At present, the only biofuels produced and used on a large scale in the world are ethanol and biodiesel. Specifically, ethanol is the most used biofuel. Data on ethanol production reveal important expansion trends. Specifically, the United States the total production of this biofuel in the year 20 11 was about 52617 million liters, showing an increase of 4.5% over the previous year. EIA (2012).

At present, the production of biofuels is mainly based on the fermentation of plant matter with high sugar content by yeasts, especially strains of Saccharomyces cerevisae. These yeasts have a great capacity for growth under fermentative conditions, tolerate high concentrations of

substrate and are highly resistant to the ethanol that they themselves produce (Sánchez, OJ, & Cardona, C. a. (2008). Trends in biotechnological production 01 fuel ethanol from different feedstocks. Bioresource Technology, 99 (13), 5270-95. doi: 1 0.1 016 / j.biortech. 2007.ll.013).

However, the use of plant raw material to obtain biofuels poses serious economic, social and environmental problems due to the necessary increase in cultivated area for this purpose. The high demand for fuels would imply the need to greatly increase the agricultural effort in order to produce enough raw material, with the economic and environmental cost that such an increase in the arable area would imply. On the other hand, the use of consumer crops for the production of biofuels implies the social and economic risk that the demand for such products affects the price of food.

To solve this problem, research in the development of biofuels is aimed at finding alternative substrates to crops. Ideally, such substrates should generate high productivity, high yield of biofuel as well as raw material, and at a competitive cost thus avoiding the use of crops. In this sense, the use of substrates derived from agroforestry, industrial or fishing waste is of great interest, assuming, in turn, an alternative treatment of them taking advantage of their potential as raw material for other processes. However, most of the available waste is not easily usable by yeasts, which makes it necessary to use pre-treatments that complicate the biofuel production process, increasing costs. Such pre-treatments usually consist of the digestion of the substrate to a state assimilable by the microorganism (eg obtaining glucose from cellulose by enzymatic digestion), and can be chemical, enzymatic or biological in nature. They are essentially a pre-digestion by other organisms in a multiphasic system.

Chitin is a 3-1,4-glucan formed by units of N-acetylglucosamine. This biopolymer is one of the most abundant on the planet, being part of the invertebrate exoskeleton, mainly arthropods, mollusks and nematodes (Rabea El, Badawy ME, Stevens CV, Smagghe G, Steurbaut W. 2003. Chitosan as antimicrobial agent: applications and mode of action Biomacromolecules, 4: 1457-65) and in the cell wall of the hyphae of true fungi. Seafood waste (mainly marine crustaceans) is an abundant source of chitin. The high nitrogen content of these wastes makes them highly eutrophizing agents. Its uncontrolled elimination causes significant environmental problems, mainly due to the accumulation of amines and other nitrogen compounds (Kandra, P., Challa, MM, & Jyothi, HKP (2012). EHicient use 01 shrimp waste: present and future trends. Applied Microbiology and Biotechnology, 93 (1),

17-29. doi: 1 0.1 007 / 500253-011 -3651 -2).

Chitin derivatives, especially partially deacetylated chitin (chitosan) possess antimicrobial activity. Nowadays, chitosan is a compound with enormous potential, from which different applications have been discovered, achieving important advances in both medicine and agriculture. Chitosan has characteristics as an antimicrobial agent due to its bactericidal and fungicidal activity, acting as a membrane destabilizing agent (Palma-Guerrero, J., Huang, l.-C., Jansson, H.-B., Salinas, J., Lopez -Llorca, L. V, & Read, ND (2009). Chitosan permeabilizes the plasma membrane and kills cells of Neurospora crassa in an energy dependent manner. Fungal Genetics and Biology, 46 (8), 585-94. Doi: 10. t01 6 / j.fgb.2009.02.010). A limitation for its use as a fuel raw material is that traditional fermenting organisms such as Saccharomyces cerevisae are sensitive to chitosan and unable to use chitin, chitosan, or even the monosaccharide components thereof (N-acetylglucosamine and glucosamine), as the only source of carbon and nitrogen. Therefore, it is necessary to design strategies for its use that reduce negative environmental impacts.

Thus, it would be interesting to have an ethanol producing agent from chitosan from waste from the seafood industry or other sources of chitin or chitosan, such that, on the one hand, a process for the production of alternative ethanol is provided. use of crops or agroforestry waste and on the other hand, it would be a way to eliminate the pollution produced by shellfish residues. In addition, the process would generate fungal biomass for agrobiotechnological use.

DESCRIPTION OF THE INVENTION

The present invention provides the solution to the problems set forth above through the use of the fungus Pochonia chlamydosporia, as an organism producing ethanol from chitosan.

Thus, in a first aspect, the present invention relates to a process for the production of bioethanol (hereinafter the process of the present invention) to

from a source of chitosan that includes the use of the fungus Pochonia chlamydosporia.

In the present invention, a source of chitosan means any product comprising chitosan, chitin or derivatives of both products.

In a particular aspect of the present invention, the source of chitosan used in the process of the present invention is chitosan, chitin or products derived therefrom.

In a particular embodiment of the present invention, the source of chitosan comes from shellfish residues.

In a particular embodiment of the present invention, the fungus is in a concentration such that it allows the minimum growth necessary to perform the process of the present invention without the phenomenon of growth self-inhibition occurring. More particularly, the fungus is found in a concentration between 104 _107 spores / ml.

In the present invention, by spores it refers to conidia, clamidospores or any type of manageable and quantifiable fungal inoculum that easily generates rapid and abundant growth of the producing species of said inoculum.

In a particular embodiment, the process of the present invention comprises the addition of nutrients to the reaction medium, such as POS (Potato Dextrose Broth).

In another aspect, the present invention relates to the use of Pochonia chlamydosporia, for the production of ethanol from chitosan.

In a third aspect, the present invention relates to the use of Pochonia chlamydosporia, for the degradation and decontamination of shellfish residues and obtaining fungal biomass and bioethanol.

The process of the present invention provided the following advantages:

one.

P. chlamydosporia, has the ability to grow in the presence of high concentrations of chitosan (more than 2 mg / ml) (Palma-Guerrero et aL, 2008) that are toxic to other organisms (eg S. cerevisiae) commonly used in the Biofuel production. This characteristic also represents an advantage by reducing the probability of contamination of the cultures of P chlamydosporia for the production of bioethanol, with other microorganisms.

2.

P. chlamydosporia has the capacity to produce a high amount of reducing sugars, especially in anaerobiosis, in the presence of high concentrations of chitosan (up to 3 mg / ml), which can be subsequently fermented to ethanol.

3.

P. chlamydosporia has the ability to grow under anaerobic conditions using chitosan as the sole source of carbon.

Four.

P. chlamydosporia, has the ability of degradation of chitosan in anaerobiosis, achieving a degradation of such a compound greater than in aerobic conditions.

5.

P. chlamydosporia, has the ability to tolerate ethanol in the culture medium.

6.

P. chlamydosporia, presents in its genome, zinc-dependent alcohol dehydrogenases and pyruvate decarboxylases necessary for the production of ethanol.

7.

P. chlamydosporia, presents the ability to produce ethanol from chitosan, an abundant residue of the seafood industry, whose main source is the exoskeletons of crustaceans, under anaerobic conditions.

DESCRIPTION OF THE FIGURES

Figure 1. It shows the growth in liquid medium of P. chlamydosporia with chitosan (O, 0.25, 0.5, 1, 2 and 3 mg mr ') as the only source of nutrients.

Figure 2. It shows the ability to degrade chylosan by P chlamydosporia with different concentrations of chitosan (0.5 and 1 mg mr ') as the sole source of nutrients in solid agar-water medium (1.5%) under aerobic and anaerobic conditions. Index 1- (C / H) represents the chitosan degrading capacity by the fungus relative to its growth by relating the diameter of the degradation halo of said substrate (H) to the diameter of the fungus colony (C).

Figure 3. Shows the appearance of the 15-day colonies of P. chlamydosporia growing on cornmeal agar with chitosan in aerobic or anaerobic conditions. Note the dark area around the colonies of the fungus that corresponds to the chitosan degradation halo.

Figure 4. It shows the production of reducory sugars by P chlamydosporia during its growth in liquid medium with 0, 0.25, 0.5, 1, 2 and 3 mg mr 'of chitosan under aerobic conditions.

Figure 5. It shows the production of reducing sugars by P chlamydosporia in liquid medium with 0, 0.25, 0.5, 1, 2 and 3 mg mr 1 of chitosan under anaerobic conditions.

Figure 6: Shows the growth of P. chlamydosporia in agar-water medium (1.5%) supplemented with alcohol (O, 1, 2, 3, 4, 5 and 10%).

Figure 7: Multiple alignment of fungal genes of alcohol dehydrogenases I with the homologs predicted in the genome of P chlamydosporia. It includes the sequences of Saccharomyces cerevisiae, Arabidopsis thaliana, Gandida albicans (2 sequences, it has a gene duplication), Neurospora crassa, Schizosaccharomyces pombe, Fusarium oxysporum, AspergiIJus terreus, Trichoderma reseei, CJaviceps purpurea, Meveriapisapisapisapisapisaaaaaaaaaaaaaaaaaaaaa.

Figure 8: Phylogenetic tree to illustrate fungal alcohol dehydrogenases including those predicted in the genome of P. chlamydosporia. The tree was constructed using the same sequences as in the alignment of Figure 7. The Arabidopsis thaliana sequences have been included as an external group in order to root the tree.

Figure 9: Multiple alignment of the amino acid sequences of the Pyrovate decarboxylases of P chlamydosporia with the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe and Gandida albicans (2 sequences, presents a gene duplication), and those of the filamentous fungi Neurospoper crassaus, Fusariumgysium, Fusariumgusium, Fusarium gilliumus, terreus, Trichoderma reseei, Glaviceps purpurea, Gordyceps militaris, Beauveria bassiana and Metarhizium anisopliae. Those of the Arabidopsis thaliana model plant have also been included.

Figure 10: Phylogenetic tree to illustrate fungal pyruvate decarboxylases including those predicted in the genome of P. chlamydosporia. The tree was constructed using the same sequences as in alignment of Figure 9. The Arabidopsis thaliana sequences have been included as an external group in order to root the tree.

Figure 11: Shows the production of ethanol by P. chlamydosporia under anaerobic conditions with chitosan (0, 0.25, 0.5, 0.75, 1, 2 and 3 mg / ml) as the only carbon source at 0, 2, 4, 6, 8, 10 and 12 days of growth in anaerobiosis.

DETAILED DESCRIPTION OF THE MODES OF EMBODIMENT

The present invention shows the ability of P.chalamydosporia to grow efficiently in anaerobiosis using chitosan as the sole source of nutrients and to produce ethanol from sugars, products of the degradation of this residue compound of the seafood industry.

The chitosan to be used in the method of the invention can be generated by chemical or enzymatic deacetylation of animal or fungal chitin.

EXAMPLE 1: Growth of P. chlamydosporia, in chilosano and degradation of this substrate as the sole source of nutrients

The growth capacity of P. chlamydosporia was analyzed using chitosan as the sole nutrient, in liquid or solid medium. The P. chlamydosporia isolate used in the embodiments of the present invention, was strain 123 (Pc123), from Seville, obtained from infected eggs of the Heterodera avenae nematode, belonging to the fungal collection of the Phytopathology Laboratory, Department of Marine Sciences and Applied Biology, University of Alicante and deposited in the CECT with the reference number 20929. The chitosan employee, named as T8s, was purchased from Marine Bioproducts GmbH (Bremerhaven, Germany). This chitosan has a molecular weight of 70 kDa and a degree of deacetylation of 82.5%. Prepared for use in the experiments detailed below, a 1% solution of chitosan in 0.25M hydrochloric acid is prepared, adjusting the pH to 5.6 with 1M sodium hydroxide. This solution is dialyzed for 3 days in distilled water at 4QC to eliminate the salts present in the chitosan or the possible salts formed in the pH adjustment process. The dissolved and dialyzed chitosan is sterilized by moist heat in autoclave before use.

The strains of P chlamydosporia, were grown in Petri dishes with corn extract agar culture medium (CMA) at a temperature of 250C and reseeded every 30 days. For the tests carried out in liquid, after 20-30 days, the conidia and clamidospores were extracted in sterile distilled water and 0.05% Tween20. The spore suspension in sterile distilled water was filtered through Miracloth and quantified in a Neubauer chamber to adjust to the final concentration of 106 conidia / ml. The treatments were: O, 0.25, 0.5, 0.75, 1, 2 and 3 mg / ml of chitosan. Once the chitosan dilutions were prepared, they were inoculated with P. chlamydosporia conidia and 12 aliquots of each treatment were loaded on a multiwell plate and the growth was estimated as the absorbance at 490 nm every 24 h for 15 days using the GeniosTM Multiwell spectrophotometer ( Tecan Mánnedorf, Switzerland) (Lopez-Moya, F., Colom-Valiente, MF, Martinez-Peinado, P., MartinezLopez, JE, Puelles, E., Sempere-Ortells, JM and Lopez-Llorca LV (2015) Carbon and

nitrogen limitation increase chitosan antifungal activity in Neurospora crassa and fungal human pathogens. Fungat Biolog and. doi: 10.1 016 / j.funbio. 2014.12.003.).

With the values obtained, the growth curves were constructed for each of the treatments, observing that P chlamydosporia was able to grow in a wide range of chitosan concentrations as the only source of nutrients. In addition, P. chlamydosporia showed a considerable growth, providing information about its high capacity to degrade chitosan and use it as the sole source of nutrients. In addition, this fungus does not require the addition of PDS (0.025%) for the germination of its conidia, so it still has a greater advantage for its use as a degradator of such a substrate (Figure 1).

Pochonia chlamydosporia is a chitosan resistant fungus, capable of degrading this compound. Growth tests of P. chlamydosporia, in solid medium, were performed on agar-water plates (1.5%) supplemented with chitosan (O, 0.5 and 1 mg / ml) and inoculated with an agar fragment with mycelium agar P. chlamydosporia using a punch. The fungal growth halo was measured daily as well as the chitosan degradation halo produced by the fungus on those plates containing this compound. These tests were carried out in order to show the differences in growth and degradation between aerobic and anaerobic conditions, therefore, this same experiment of fungal growth and degradation of chitosan in plaque was performed under anaerobic conditions, taking the measures every 4 days To achieve such conditions, the plates were placed in anaerobic jugs (Oxoid) using the anaerobic atmosphere generation system wAnaeroGen "(Oxoid). The growth capacity of P ch / amydosporia was observed, in anaerobiosis and the degradation of chitosan that was greater than that observed in aerobic conditions ..

EXAMPLE 2: Production of reducing sugars by P. chlamydosporia, in the presence of chitosan

In order to measure the production of reducing sugars by P. chlamydosporia, in the presence of different concentrations of chitosan, 100 ml flasks were prepared with 20 ml of chitosan dissolved in water at the following concentrations: 0, 0.25, 0.5, 0.75, 1, 2 and 3 mg / ml. In these flasks the fungus was inoculated using the extracted conidia (as in Example 1) at a final concentration of 106 conidia / ml. These flasks were incubated with shaking (150 rpm) at 252C for 15 days. Samples were taken every 24 hours from the cultures and the amount of sugars was measured

Reducers using Dinitrosalicylic Acid (ONS) (G. L. Miller (1959) Use of Dinitrosalicylic Acid Reagent lar Determination 01 Reducing Sugar, Anal. Chem. doi: 10.1021 / ac60147a030). For this, 1 ml of sample, 1 ml of 11% ONS solution was added and incubated for 10

min at 90 !! C, then 330 1-11 potassium tartrate (40%) was added and after cooling the sample to room temperature the absorbance reading was read at 595 nm using the GeniosTM Multiwell spectrophotometer (Tecan Mannedorf, Switzerland). The results of production of reducing sugars are shown in Figure 4, showing that their production is increased in the presence of high concentrations of chitosan, especially at concentrations of 2 and 3 mg / ml. Similarly, the production of reducing sugars under anaerobiosis conditions was measured, using the same protocol described, taking the samples, which in this case were incubated in the anaerobiosis jugs, every 2 days. In Figure 5, an increase in the production of reducing sugars under anaerobic conditions at high concentrations of chitosan is observed, although less than that observed at such concentrations in aerobic conditions.

EXAMPLE 3: Growth of P. chlamydosporia, in the presence of ethanol.

Since one of the requirements of an ethanol producing organism is to tolerate the presence of ethanol in the medium, an experiment was conducted to see the fungal growth in 1.5% agar-water supplemented ethanol (O, 1, 2, 3, 4 , 5 AND 10%). The medium with ethanol was inoculated with P. chJamydosporia, as in Example 1, using a punch. Fungus growth was measured daily and growth curves were constructed (Figure 6). P. chlamydosporia is able to grow to alcohol concentrations of 4%, showing radial growth in medium with ethanol.

EXAMPLE 4: Comparison of the sequences of the Alcohol dehydrogenase (ADHs) and Pyruvate decarboxylases (PDCs) genes of P. chlamydosporia, compared to those of other organisms capable of producing ethanol.

The sequences of various alcohol dehydrogenases I and pyruvate decarboxylases described were aligned by multiple alignment with the homologous genes predicted in the genome of P. chlamydosporia (Larriba, E., Jaime, MD L, Carbonell-Caballero, J., Conesa, A., Dopazo, J., Nislow, C., Lopez-Llorca, L V. (2014). Sequencing and functional analysis of the genome of a nematode egg-parasitic fungus, Pochonia chlamydosporia. Fungal Genetics and Biology, 65, 69 -80. Doi: 10.1016 / j.fgb. 2014.02.002). The alignment (Figure 7) included the sequences of Saccharomyces cerevisiae, Arabidopsis thaliana, Gandida albicans, Neurospora crassa, Schizosaccharomyces pombe, Fusarium oxysporium, Aspergillus terreus, Trichoderma reseei, Claviceps purpurea, Cordycepialiais beetis. The alignment was done using T-Coffee. With respect to the alignment of alcohol dehydrogenases (Figure 7), the protein Pc_10387 has a high percentage of identity with similar fungal sequences, so we conclude it is the orthologous gene. Pc_ 10376 has a lower identity percentage than expected with nearby fungi and seems to lack a region of about 60 amino acids at the N-terminal end. Due to the percentage of identity that it has with the sequences of yeasts and other fungi, it is probable that it is a duplicated gene and that it possesses the same enzymatic activity. However, some degree of subfunctionalization similar to that observed in Saccharomyces cerevisiae or Gandida albicans cannot be ruled out. The alignment of the pyruvate carboxylase sequences are presented in Fig. 9. The sequence has a high percentage of identity with nearby fungi, forming a monophyletic cling with the sequences of Metarhizium anisoplae and Glaviceps purpurea. It is surprising, however, the position of the sequences of Beauveria bassianna and Cordyceps militaris. The protein has a high degree of general preservation, although there are at least three variable regions in its sequence.

The tree was constructed by aligning the sequences with MAFFT v.7.164. Subsequently, the alignment was cleared using Gblocks v 0.91 b. The tree was constructed using a Neighbor Joining algorithm, a JTT replacement model and a 100 bootstrap (Figure 8). Pc_10376 is noted as alcohol dehydrogenase 1, ortholog of alcohol dehydrogenase (AOH) involved in the production of ethanol in yeast. Pc_10367 has a similar percentage of identity with yeast, but relatively low with ADHs of filamentous fungi near P chlamydosporia. Pc_10376 appears in our phylogenetic tree as a sister group of the canonical ADH1 of Metarhizium anisopliae, forming a sibling brother to the ortholog of Trichoderma reesei. Pc_10367, on the other hand, appears as the sister group of the ADH of Aspergillus terreus. Although the results are inconsistent with the established taxonomy of these groups, the phylogenetic distances and bootstrap supports indicate that ADH is not a good marker to solve the phylogeny of these groups, and we must consider that the sequences do not differ enough to adequately solve phylogenetic relationships. In view of these results, we conclude that Pc_10367 is the result of a gene duplication with subsequent subfunctionalization (paralogue) or a horizontal transfer event (xenologist). It would be necessary to address more comprehensive evolutionary studies to rule out or support either of the two hypotheses. Like this gene, it is not present in other parasitic fungi of nearby ecdisozoans, we consider that Pochonia chlamydosporia has "extra" resources for the production of ethanol. Several genes of the P chlamydosporia genome are noted as pyruvate decarboxylase, but because the homology search was performed, only the Pc_2054 gene was chosen for the construction of the tree (Figure 10).

EXAMPLE 5: Production of ethanol by P. chlamydosporia, in the presence of chitosan.

In order to check the production of ethanol from P chlamydosporia under anaerobic conditions, Pyridinium Chlorochromate (PCC) was used. The PCC is the reagent most used in the quantification of ethanol by determining its oxidation to carbonyl groups. 10 ml aliquots of the 5-day aerobic cultures of P chlamydosporia were obtained, with chitosan (0-3 mg / ml) as a single nutrient source, they were transferred to anaerobic conditions in 4.7 cm diameter Petri dishes in the jugs of anaerobiosis. These cultures were kept under gentle agitation (100 rpm) and samples were taken at 0, 2, 4, 6, 8, 10 and 12 days to measure ethanol production in them, with time O, the value before incubate the cultures in an anaerobic environment.

PCC measurements were made by extracting a 004 ml aliquot from the culture broth that was incubated with 0.4 ml of 1 M PCC for 10 min at room temperature. The samples were centrifuged at 10,000 rpm for 10 min and the absorbance at 570 nm of the supernatant was measured. The absorbance values obtained were transformed into% ethanol using a calibration line made with increasing concentrations of ethanol (Abs570 = 0.0457 *% ethanol + 0.0116) obtaining an adjustment of R2 = 0.9974. As shown in Figure 11, P chlamydosporia produces ethanol from day 4, the production values obtained are between 0.5 and 2%, in cultures with 1-3 mglml of chitosan.

Claims (6)

1. Procedure for the production of bioethanol from a chitosan source comprising the use of the fungus Pochonia chlamydosporia, 2. A method according to claim 1, wherein the source of chitosan is a product comprising chitosan, chitin or derivatives thereof.

3.

Method according to any of the preceding claims wherein the source of chitosan comes from shellfish residues.

Four.

Method according to any of the preceding claims, wherein the fungus is in a concentration comprised of 104_107 spores / ml.

Method according to any of the preceding claims, which includes the addition of nutrients.

6.

Use of Pochonia chlamydosporia, for the production of ethanol from chitosan.

7.

Use of Pochonia chlamydosporia, for the degradation of shellfish residues and

obtaining fungal biomass and bioethanol. fifteen