ES2329643B1 - PSEUDOMONAS SP. PME 707 (CECT 7314) GRINDER OF OILY WASTE FROM THE FOOD AND HOTEL INDUSTRY, AND PROCEDURE FOR APPLICATION. - Google Patents

Abstract

Pseudomonas sp Pme 707 (CECT 7314) degrader of oily waste from the food and hospitality industry, and procedure for its application.

Bacterial strain Pseudomonas sp Pme 707 (CECT
7314), characterized by its degrading capacity of the aqueous-oily fraction of the oily residues of the food and hospitality industry, due to its stimulating capacity of the microbial activity by quorum sensing and of the plant growth for compost formation, and its capacity to biotransformation of fats by their enzymatic activities esterase, esterase lipase and lipase. This strain has been isolated and characterized to be used in the biological treatment of the oily effluents components of said waste, classified as toxic or dangerous, generated during the process of making olive oil and other vegetable oils, as well as after its subsequent consumption , according to a procedure, which for the particular case of emulsions of edible oils used, consists of a prior physical-chemical treatment of the residue and the subsequent inoculation of the microorganism for biotransformation.

Description

Pseudomonas sp Pme 707 (CECT 7314) degrader of oily waste from the food and hospitality industry, and procedure for its application.

The invention presented below is aimed at the bacterial strain Pseudomonas sp , of laboratory code Pme 707, isolated and characterized for the purpose of being used in the biodegradation of oily waste from the food and hospitality industry, as is the case of the manufacture and consumption of olive oil, due to the ability of said microorganism to favor microbial growth mediated by quorum sensing and plant growth by production of auxins in the aqueous-oily fraction of said waste, classified as toxic or dangerous, as well as its ability to transform the fats there for its enzymatic activities esterase, esterase lipase and lipase.

This strain has been deposited for the purpose of patent in the Spanish Type Culture Collection (CECT), dated October 16, 2007, where it has been assigned the number 7314. The CECT is based in the research building of the University of Valencia, located on the Burjassot campus (DP 46100 - Valencia Spain).

The object of the present invention is also the procedure by which the microorganism is applied to treatment of those oily residues from consumption in hospitality of vegetable oil, mainly olive oil, with the so that the degradation process of oily emulsions can really take place.

The invention falls within the field of biotechnology, and within it, in the biological treatment of effluents and oily waters components of waste from Toxic or dangerous oils, both from the food industry, generated during the manufacturing process (known alpechines and alpeorujos in the case of olive oil), such as produced after consumption in hospitality (oil emulsions used groceries).

For being a method of biological treatment of waste, is in itself an environmentally friendly method and economical, compared to traditional disposal or of physical-chemical treatments of waste oily, being in this case especially interesting in how much it favors the transformation of said waste into compost, due to the ability of the microorganism to produce auxins that They favor plant growth.

State of the art

Waste is defined as any substance or object from which its holder detaches or intends or obligation to detach (Law 10/1998 on waste). In agreement under current legislation, hazardous wastes are those that they meet at least one of the following characteristics: explosiveness, oxidizing, flammable, irritating, harmful, toxic, carcinogenic, corrosive, infectious, teratogenic, mutagenic, unstable, source of secondary or eco-toxic compounds. How example we can say that a residue is corrosive if it presents any of the following properties: be aqueous and have a pH less than 2 or more than 12.5, and / or be liquid and corrode the steel to a higher rate of 6.35 mm / year at a temperature of 55 ° C. And a residue would be toxic when it has the potential to cause death, serious injuries, or detrimental effects on the health of the being human if ingested, inhaled or comes into contact with the skin.

As a result of certain activities related to vegetable oils, such as manufacture of olive oil, the hospitality sector, or the pig production, oily effluents are generated that include substances toxic to health and harmful to the environment environment, so they are considered toxic waste or dangerous.

In the food and hospitality industry said existing substances as a result of many processes related to vegetable oil, as is the case of olive oil. In the effluents of oil production of olive, both in oily waters or alpechines (method of extraction continues in three phases), as in the pasty alpeorujos (two-phase extraction method), there is a high concentration of organic matter and different inorganic compounds, such as case of polyphenols, polyalcohols, nitrogen substances, etc., all of a very toxic nature and in turn very diverse. If Particular of polyphenols, most are considered extremely toxic to animal cells and contaminants for the environment, they also produce an eutrophication action very accused of the waters, which justifies the enormous effect waste contaminant.

Therefore, the last fraction of waste from the production and consumption of vegetable oils, formed by oily waters or alpechines, on which this proposal of patent are considered as hazardous waste, both by the referred state legislation (Law 10/98 of Waste), as per the Community (Council Directive 91/156). In the directive of the EU of hazardous waste 91/689 / EEC in its Annexes I, II and III, classified into generic categories or types of hazardous waste related according to their nature or the activity that generated them. Thus, we have in Annex I.A. nº 9.- oil / water, mixtures of hydrocarbon / water, emulsions; or in annex I.B. No. 19.- soaps, animal or vegetable fats and waxes; nº 38.- vegetable oils. We can also check this classification in the European Catalog of Waste (CER), included in chapter 20 of the list, corresponding to municipal waste (household waste and similar from commercial industries and institutions), including selectively collected fractions at point 20 01 25 and 20 01 26.

That is why the Waste Law obliges producers to eliminate (or transform) waste oily, either through an authorized manager, or through a treatment, such as an WWTP in the case of production of porcine. Its removal is a real problem for certain companies, especially if they are included in Annex 1 of the IPPC, as they are required to obtain Authorization Integrated Environmental

Traditional methods for elimination or treatment of effluents from the production of vegetable oils, especially regarding oily waters rich in polyphenols, have been direct elimination, through treatment plants, fertilizers, feed or controlled spills, the evaporation, natural in rafts or accelerated by heat treatment, and physical-chemical treatments (filtration, incineration, distillation-evaporation, etc.). These methods are generally harmful to the environment, and Also quite expensive. That is why, since a few years ago, they have severely disrupted biological treatment methods, such as the generation of biogas, composting or fermentation, which is in the field where the invention falls below presents.

Legislation and technology oriented to the cleanliness of the environment and the prevention of its deterioration have been two of the greatest advances of the end of the twentieth century and have led to birth of technologies based on the use of microorganisms with broad biodegrading capabilities.

One such technique based on the use of microorganisms is bioremediation, which uses microorganisms live for the removal of environmental pollutants (Atlas and Pramer, 1990; Atlas and Bartha 2002). This is one of the technologies. applicable to the purpose pursued with the present invention, even to knowing that in the case of oily effluents, the toxic or dangerous components generally maintain a low degradation rate, in addition to only a few species of microorganisms, hence they are more difficult to eliminate naturally and persist for longer affecting the environment ambient.

However, in the Mediterranean area, the degrading activity of oily emulsions may result especially interesting. This activity is not developed in general by gram-negative bacteria, but the proliferation of these can facilitate the production of a biocompost from a residue.

What has been achieved in this case is to isolate a bacterial strain capable of stimulating the microbial growth of other groups of degrading bacteria of toxic and dangerous components of oily effluents of origin in the food and hospitality industry, and of producing substances that favor plant germination, favoring the transformation of waste into biocompost. In addition, the new microorganism has enzymatic activities esterase, esterase lipase and lipase that allows it to directly degrade the fats of these
effluents

But said microorganism potentially Oil residue degrader cannot be added directly to the residue, but requires a treatment previous physical-chemical of the same for use, which is also the object of this invention.

There is currently no work on the valuation of the last fraction aqueous-oily in toxic oils of origin industrial or hospitality to obtain compost, published in patent documents or through non-patent literature, it It is appropriate to propose the present elimination alternative or reuse more environmentally friendly than employed so far.

The invention

The aforementioned potentially degrading oily residue microorganism of the food and hospitality industry, and which is claimed in its own invention, is Pseudomonas sp Pme 707, with deposit number 7314 in the Spanish Type Culture Collection (CECT) for patent purposes.

It is a microorganism capable of produce substances that stimulate mediated microbial activity by quorum sensing of different bacteria that help the degradation of the different residual components of the waters and oily effluents generated during the production process of vegetable oils, and which are also present in emulsions of used oils from large-scale consumption as well as per the metabolic capacity it has for the biotransformation of existing fats in said fraction aqueous-oily toxic residue originating in the Industry and hospitality.

More specifically, the degrading power of wastewater and oily effluents of Pseudomonas sp Pme 707 due to stimulation of microbial activity is due to the ability of this microorganism to produce molecules with signaling function, which diffuse into the organic matter of the components of said residues, so that when reaching a sufficient concentration or critical density, it is caused by the aforementioned quorum sensing phenomenon, translated as "perception of the medium" or "self-induction", that different types of bacteria existing in the medium recognize said concentration of organic matter, modulating part of its gene expression and accentuating its microbial activity against these residual components, causing its degradation.

The microorganism in question also presents an important production capacity of auxins that favor the germination and plant growth, which facilitates the biotransformation of the residue in compost.

Once isolated and characterized, it has verified as the strain Pme 707 is especially interesting for the biotransformation of oily residues rich in substances toxic origin in the food and hospitality industry. Between Its characteristics include the following:

It is a mesophilic microorganism, so the ideal incubation temperature should be between 28 and 40 degrees Celsius.

Maintains an aerobic metabolism.

It behaves like a neutrophil and maintains a rate of optimal biotransformation in pH from 5.5 to 7.5.

It has a fat-degrading ability through activities esterase, esterase lipase and lipase.

In cases where the oily residue of character  toxic or dangerous are made up of used vegetable oil emulsions (oily water with dissolved ionic charge), coming mostly volume of hospitality consumption, a Method of application of the bacterial strain of the invention for enable its biodegrading action of polyphenols and others toxic components once in contact with the residue.

This procedure, which is also claimed as of own invention, it consists of a treatment physical-chemical prior to waste and subsequent inoculation of the microorganism for biotransformation, according to these three basic operations described below in detail:

1) Emulsion of residual oil in decanter and separation of the aqueous-oily phase from the organic by water vapor flotation, performed by injection of water vapor from the bottom of the decanter.

2) Phase pH correction aqueous-oily up to a value between 5 and 7.5.

3) Inoculation of the microorganism in the phase aqueous-oily to cause its biotransformation

The advantage of this invention is to have a bacterial strain potentially degrading components Toxic or dangerous present in water and oily effluents generated during the production and consumption of vegetable oils, as olive oil, as it stimulates microbial activity of various bacteria that carry out such degradation and acts directly on different types of fats in the themselves, being also a strain especially beneficial for environment, as it facilitates the biotransformation of waste in compost, due to its auxin production capacity that favor germination and plant growth.

In the case of waste treatment of used vegetable oils, which are a problem in the sector of hospitality, the application procedure for the strain in question to the aqueous-oily phase of the oily emulsions, which makes the invention can be directly usable in the industry.

The graph in Figure 1 reflects the stimulating capacity of the quorum sensing mediated microbial activity of the Pseudomonas strain, assessing the production of violacein by the mutant strain of Chromobacterium violaceum CV026 after a 24-48 hour incubation period in culture with extract of microbial by-products of Pseudomonas sp Pme 707.

Application mode

The methodology for applying the strain Pseudomonas sp Pme 707 to oily residues of a toxic or dangerous nature has already been outlined above for a case where that residue is composed of emulsions of used vegetable oil (oily water with dissolved ionic charge) mainly from consumption in the hospitality industry.

As noted, the first is the preparation of the residue by treatment physicochemical, according to a process in which said emulsions are subjected to a steam flotation stage of water in order to separate the organic phase from the phase aqueous-oily, followed by the pH correction of This second.

The flotation operation is performed in a decanter in which steam is introduced through ducts with numerous injection nozzles 0.5 mm in diameter.

Water vapor injection from the bottom of the  decanter produces three effects: The first heating of the emulsion, thereby separating the organic phase from the aqueous one by increasing the buoyancy of oil with respect to water, the second it is the drag by flotation of the oil towards the surface, produced by the small ascending bubbles of steam, and by Finally, the solubilization capacity of acids is increased organic, ketones and aldehydes in the aqueous phase.

The organic phase can be used for others purposes such as biodiesel production, not being tested for The present invention. However, the phase aqueous-oily does not present this utility, being interesting to biotreat it with the intention of detoxifying it and being able to value it as compost or other uses.

The pH of the aqueous oil phase of the residue must be corrected until neutrality is approached before of the inoculation of the microorganism for its biotransformation. For that the process is optimal, the pH should be maintained between 5 to 7.5.

For pH correction it is proposed to use NaOH (sodium hydroxide) in concentration and volume according to amount and composition of the residue.

Once the residue is prepared, the inoculum of the Bacterial strain could be prepared from any general means complete and complex. The volume and concentration of the bacteria should adapt to the volume of the waste to be biotransformed, as well as the Bi-reactor to be used.

Treatment with this microorganism looks favored if periodic oxygenation of the substrate occurs biotransform. This improves the germination rate vegetable.

Inoculation of the microorganism in the phase aqueous-oily is made from a culture previous in liquid medium LB at 37 ° C for 24 h, until reaching a concentration greater than 5 x 10 11 cfu / ml (forming units of colonies per milliliter), verified by the counting method in plate, and inoculated at the rate of 500 ml of culture per cubic meter of residue.

Examples

The results of some of the tests and experiments that justify the efficacy of the invention

Stimulating capacity of microbial activity mediated by quorum sensing

A detection of producing activity of quorum-sensing activating molecules was carried out by means of the biological test in the model of Chromobacterium violaceum (McLean RJ et al , 2004). The assay consists of detecting the production of violacein by the mutant strain of Chromobacterium violaceum CV026 after an incubation period of 24-48 hours. The following steps were followed:

1. The strain of Pseudomonas sp was grown in a complete medium for 24, 48 and 72 hours.

2. The growth extract was filtered by a 0.22 µm filter leaving an extract of the medium eluted without bacteria

3. The mutant strain of Chromobacterium violaceum CV026 was inoculated into the extract and incubated for 24, 48 and 72 hours.

4. The activity was assessed as positive quorum-sensing regulator if the Violacein production.

5. The absorbance of the extracts at 558 nm was measured to quantify the production of violacein by the strain of Chromobacterium violaceum CV026 in culture with extract of microbial by-products of Pseudomonas sp Pme 707 CECT 7314 under different conditions.

Table I shows the evaluation of the production of violacein by the strain of Chromobacterium violaceum CV026 in culture with extract of microbial by-products of Pseudomonas sp Pme 707 CECT 7314 at 0, 24 and 48 hours, being Absorbance of the production of violacein in full culture and B the absorbance in culture with extract of microbial by-products of Pseudomonas sp Pme 707. The graph in Figure 1 reflects the result of this assessment.

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TABLE I

one

Fat biotransformation capacity

Table II shows the assessment of the microbial activity esterase, esterase-lipase and lipase at 24 hours according to the ZYM BioMèrieux API method.

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TABLE II

2

Auxin production capacity

Indolacetic Acid (AIA) is an auxin, it is that is, a plant hormone that favors the growth of plants. AIA production was estimated using the method of colorimetric assessment of Gordon and Weber, (1951).

After the colorimetric evaluation, the technique described by García de los Ríos, 1989 was used and after its methodology it was found that the maximum value detected for the strain of Pseudomonas sp . Pme 707 CECT 7314 proposed for a patent was 2.7 µg / mL at 120 h.

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Preparation of aliquots and germination test

Homogenized 100 mL of oil and 900 mL of Water in a 1L Erlenmeyer we follow two routes:

We take 100 mL of that prepared medium, you add 1.5 g of noble agar, correct the pH up to 7, autoclave sterilized (120ºC, 20 minutes), and finally we dispense in a petri dish, obtaining plate A.

The remaining 900 mL of oil and water are autoclaved (120 ° C, 20 minutes), then added to the  Total 13.5 g of noble agar, 18 g of triptone water and bacteria corresponding control for each plate. Once sterilized (120 ° C, 20 minutes) is dispensed on a Petri dish and number

Table III shows the assessment of the germination capacity of Arabidopsis thaliana (Co10) on different substrates

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TABLE III

3

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For the germination test, 10 seeds of the Arabidopsis thaliana (Co10) species were placed on each plate and allowed to stand for 4 days under optimal conditions of temperature (approx. 24 ° C) and humidity for growth.

A last reuse of this waste (recovery) could be the biocompost being this strain a good candidate for this biotransformation for being present in a way normal on free ground. These microorganisms can favor the development and protection of crop species.

The proposed patent strain shows a marked activity esterase and esterase-lipase. From this way the fractionation of fats can facilitate the Atmospheric oxygen dissolution of the aerobic microbiota. This It could be one of the keys that regulate the effectiveness of the biotransformation in this process.

Generally, studies directed towards biodegradation have been carried out under anaerobic conditions in bioremediation processes (Bouallagui et al ., 2004), such as the biodegradation of vegetable oils and their metabolic intermediates in oil-enriched freshwater sediments (Li et al ., 2005), but not exclusively. According to Eusebio et al ., (2005), our study has been carried out under aerobic conditions, which may generate alternatives to the different biodegradation methods, especially those used with treated effluents from the agri-food industry.

Claims (6)

1. Pseudomonas sp Pme 707 (CECT 7314), a microorganism characterized by its degrading capacity of the aqueous-oily fraction of the oily residues of the food and hospitality industry, due 1) to its ability to produce substances that stimulate microbial activity mediated by quorum sensing of different bacteria that help the degradation of the residual components of the waters and oily effluents, when modulating part of their gene expression occurs at the moment of recognizing a critical concentration (the "quorum sensing" or "quorum perception"") of the molecules of said substances diffused in the organic matter of the residual components, 2) to their ability to produce auxins that favor germination and plant growth, and 3) to their ability to biotransform the fats existing in said fraction aqueous-oily 2. Use of Pseudomonas sp Pme 707 (CECT 7314), according to claims 1, in the biodegradation of the oily-aqueous fraction of oily residues of the food and hospitality industry. 3. Use of Pseudomonas sp Pme 707 (CECT 7314), according to claim 2, in the elimination of fats by means of their enzymatic activity esterase, esterase lipase and lipase from the oily waters of the food and hospitality industry. 4. Procedure for the application of the microorganism Pseudomonas sp Pme 707 (CECT 7314) to toxic or dangerous residues of used vegetable oils, for the purposes of the preceding claims, consisting of the following operations: 1) emulsion of oil in decanter and separation of the aqueous-oily phase of the organic by means of water vapor flotation, performed by injection of water vapor from the bottom of the decanter, 2) correction of the pH of the aqueous-oily phase to a value between 5 and 7.5, and 3) inoculation of the microorganism for biotransformation of the residue. 5. Procedure for the application of the microorganism Pseudomonas sp Pme 707 (CECT 7314) to toxic or dangerous residues of used vegetable oils, according to claim 5, characterized in that the correction of the pH of the aqueous-oily phase is carried out by the addition of sodium hydroxide ( NaOH), in concentration and variable volume depending on the quantity and composition of the residue. 6. Procedure for the application of the microorganism Pseudomonas sp Pme 707 (CECT 7314) to toxic or dangerous residues of used vegetable oils, according to claim 5, characterized in that the inoculation of the microorganism in the aqueous-oily phase is carried out from a previous culture in liquid medium LB at 37 ° C for 24 h, until reaching a concentration greater than 5 x 10 11 cfu / ml (colony forming units per milliliter), verified by the plate count method, and inoculated at a rate of 500 ml of culture per cubic meter of residue.