ES2470819A1 - Test and procedure for the evaluation of the rapid biodegradability of water by pseudomonas putida (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Test and procedure for the evaluation of the rapid biodegradability of waters by pseudomonas putida. The invention consists of a new kit and procedure for evaluating the "rapid biodegradability" of contaminated water. The test is based on the use of capsules containing an adequate proportion of lyophilized pseudomonas putida bacteria and mineral salts that are added to water whose biodegradability is to be evaluated. During three days of incubation at 30º c, the chemical oxygen demand (cod) is monitored and the biodegradability efficiency is calculated at the end of the test. This test is aimed at the sector of the treatment of aqueous effluents, especially of industrial origin to track the biodegradability. The method solves the problems of repeatability and testing time of traditional methods. (Machine-translation by Google Translate, not legally binding)

Description

TESTING AND PROCEDURE FOR THE EVALUATION OF WATER BIODEGRADABILITY BY WATER PSEUDOMONAS PUTlDA.

Technical sector

The present invention is directed to the aqueous effluent treatment sector,
especially of industrial origin.

GENERAL DESCRIPTION OF THE INVENTION

The invention consists of a new kit and a new method of evaluating the "rapid biodegradability" of contaminated water. This kit consists of freeze-dried Pseudomonas putida bacteria capsules and mineral salts that are added to the water whose biodegradability you want to evaluate. The Chemical Oxygen Demand (COD) is measured and the biodegradability efficiency is calculated three days after incubation at 30� (,

The kit is applied to the monitoring of the biodegradability of aqueous effluents and for the control of influencers at the Industrial Wastewater Treatment Plant (EDARI). The alternatives to evaluate the biodegradability in waters proposed by the OECD and ISO (Zahn-Wellens, BOD / COD ratio, respirometry, etc.) are based on the use of active sludge and present numerous inconveniences as to its applicability. This method solves the problems of repeatability and test time of traditional methods.

State of the art

In recent decades, the amount of organic compounds that persist in the environment has been considerably increased. In parallel, the scientific community has been developing methods to investigate and control biodegradation processes and provide adequate knowledge for environmental protection decision making. Most of the efforts have focused on the biodegradation of organic compounds in the aquatic environment, mainly in the processes of wastewater treatment, as it is a key parameter for estimating long-term risks in biota. These methods should preferably be carried out with a minimum cost, so a preliminary way to evaluate which products are dangerous for the environment is to make a simple study of their biodegradability.

The most widely used biodegradability tests are those proposed by the Organization for Economic Cooperation and Development (OECD). These tests are based on other techniques standardized by the International Organization for Standardization (ISO) or by the United States Environmental Protection Agency (EPA), among others.

According to the OECD (OECD Cuidelines for the Testing of Chemicals, Paris, France (1992)), aerobic biodegradability must first be studied by means of a "rapid biodegradability" test and if the result is negative, biodegradability can be determined by simulation test Alternatively, a test can be performed to detect "inherent biodegradability" since "rapid biodegradability" tests are more rigorous. In all cases, this organization advises that the tests be preferably carried out under realistic conditions to simulate the behavior of a particular environment (eg wastewater treatment plant (DAR), surface water, soil or sediment).

The OECD (OECD Cuidelines for the Testing of Chemicals, Paris, France (1992)) tests that can be used to determine the "rapid biodegradability" of organic substances or wastewater are the OECD No. 301 AF tests: Loss of Dissolved Organic Carbon (COD) (TC 301 A), CO2 Evolution Test (TC 301 B), Modified MITI Test (1) (TC 301 C), Closed Bottle Test (TC 301 D), Modified OECD Selection Test (TC 301 E) YTest of manometric respirometry (TC 301 F).

The "rapid biodegradability" tests are carried out under aerobic conditions using high concentrations of COD (2-100 mg COD / U of the substance whose biodegradability is being studied as the sole source of carbon (except the carbon source associated with biomass) Contaminated water is incubated for 28 days with a relatively low concentration of microorganisms (104-108 CFU / mU not adapted to the contaminant. The biomass comes from active sludge, secondary effluents from the treatment plant, soil extract

or surface water to which a specific mineral medium is added. Incubation is maintained under aerobic conditions at temperatures between 20 and 25 ° C and at neutral pH. The period can be increased above 28 days if the degradation has begun but not

It has stabilized (OECD Cuidelines for the Testing of Chemicals, Paris, France (1992)).

The "rapid biodegradability" tests are very versatile tests that can be used for a large number of contaminants because the biodegradability measurement is performed by monitoring global parameters. An example of a global parameter is the COD, which is proportional to the concentration of organic compounds present and is limited to describing the total oxidation remaining unchanged when there is no oxidation or it is very small. Other parameters are related to microbial respiration such as the Biological Oxygen Demand (BOD) or the production of carbon dioxide that indirectly provide data on the degradation of the

20 contaminant

The compound is considered to be rapidly biodegradable if 70% of the COD (TC 301 AY TC 301 E), 60% of theoretical carbon dioxide (ThC02) (TC 301 B) or 60% of the demand is eliminated of theoretical oxygen (ThDO) (TC 301 C, TC 301 OY TC 301 25 F). These biodegradation limit levels must be reached in a "10-day window" during the 28 day duration of the test (except in the TC 301 C test). The "10-day window" begins when the degree of biodegradation has reached 10% removal of COD, ThDO or ThC02 • If the result is positive it can be considered as indicative that the contaminant will be rapidly biodegraded in most

30 environments, including WWTP (OECD Cuide lines for the Testing of Chemicals, Paris, France (1992)).

The evolution of a chemical compound in an WWTP can also be studied in the laboratory using simulation tests of aerobic wastewater treatment (OECD, 1992): Active sludge unit (TG 303 A) and Biofilms (TG 303 B). For this, changes in the COD and / or COD are monitored for a recommended initial concentration of between 10 and 20 mg / L. However, many compounds usually present at lower concentrations, so more sophisticated analytical techniques are necessary. These simulation tests offer biodegradation data under specific environmental conditions through the use of native biomass and solids (active sludge, sediments, soils, etc.) that allow the adsorption of the contaminant at the typical temperature represented by the particular environment.

The "inherent biodegradability" tests have a high degradability capacity. They allow prolonged exposure times of the substance to microorganisms, a low evaluated substance / biomass ratio is used and an additional carbon source is normally added, so they offer more possibilities of obtaining a positive result than the "rapid biodegradability" tests . Some of these tests even use adapted microorganisms to increase the degree of biodegradation. Therefore, if the result is positive, it cannot be assumed that the test substance will rapidly degrade in the environment.

The most commonly used inherent biodegradability methods are tests 302 A (modified SCAS), 302 B (Zahn-Wellens / EMPA) and 302 C (modified MITI 11) (OECD, 1992). Of all the tests, 302 A has a greater capacity for biodegradation with a test time of up to 6 months, in which the substance to be evaluated (20mg COD / U is biodegraded by active sludge using a domestic effluent as a source of additional carbon The Zahn-Wellens test is carried out following the consumption of COD for 28 days and using a high concentration of test substance (50400mgCOD / U. In the modified MITI test 11 (the one with the lowest biodegradation capacity) is exposed to the contaminant to microorganisms from a mixture of natural samples of surface water, active sludge, soil, etc. for 30 days In all cases the biomass concentrations are much higher than in the "rapid biodegradability" tests (of the order 1 g STS (total suspended solids) / U.

5 A biodegradation above 20% of theory (COO, OQO or OBO) is considered as evidence of "primary inherent biodegradability" while above 70% "final inherent biodegradability". If this level is almost reached (eg ThOO or OQO slightly below 60 or 70% respectively) it is also indicative of "inherent biodegradability". Likewise, this approach applies in the event that

1O the percentage is reached but not in the "1O day window". If the result is positive, the substance has a potential degradation under favorable conditions (eg a well-operated EOAR) and if it is negative it must be concluded that the substance will be persistent in the environment.

15 Another alternative method to those proposed by the OCOE and which is widely used by various researchers to study the evolution of biodegradability is the standard test of the OBO UP Scott and OF Ollis, Journal of Advanced Oxidation Technology 2: 374-381 (1997 )). Changes in biodegradability are evaluated by measuring the OBO / OQO and / or OBO / COO ratios and comparing their values in the treated samples with those of

20 the original effluents (D. Mantzavinos and E. Psillakis, Journal of Chem. Technol. Biotechnol.,

79: 431-454 (2004)). These relationships offer an index of the proportion of organic matter present that is aerobically degradable within a certain period of time (eg 5 days in the OBOs). The limitations of the OBO test arise mainly from the fact that the velocity Biodegradation varies with concentration. Thus, the results of the

25 OBO should be interpreted with caution and should not be compared with data from other sources. In addition, OBO / OQO (or OBO / COO) relationships alone can lead to error unless individual OBO values are also given.

According to Pagga (U. Pagga, Chemosphere, 35: 2953-2972 (1997)) it is difficult to define a "method

30 true "measure of biodegradability since various environmental factors or situations may influence the results obtained. Various authors (U. Pagga, Chemosphere, 35: 2953-2972 (1997); Jiang et al., Chemosphere 48: 133-138 (2002)) suggest that the reproducibility of biodegradability measures using different methods or conditions, or even using the same test but different inocula, can generate inconsistent and sometimes even contradictory results. which most affects the reproducibility of the trials especially when its concentration is low (in the "rapid biodegradability" trials). Blok and Booy U. Blok and M. Booy, Ecotoxicology and Environmental Safety 8: 410-422 (1984 )) showed that the differences found when comparing the biodegradation of 29 chemical compounds using various methods proposed by the OECD were due to variability in the inoculum.

Reuschenbach et al. (P. Reuschenbach et al., WaterResearch 37: 1571-1582 (2003)) used two different respirometric methods to assess the biodegradability of 10 chemical compounds according to the OECD 301 F test (Oxitop� and Sapromat�). In 8 cases they obtained comparable results but in the remaining two the results were different, so they concluded that both systems were not equivalent. Likewise, K�mmerer et al. (K. K�mmerer et al., WaterResearch: 38 2111-2116 (2004)) found that the inhibitory concentrations of various antibiotics, disinfectants and cytotoxics depended strongly on the time of the test and the type of compound, concluding that the test Standardized OECD used failed to study the environmental effects of these organic compounds.

The OECD indicates that standardization of inoculum can improve the comparison of results between different trials. However, it is difficult to standardize the inoculum if at the same time a large number of species is required in the test, since this organization recommends a mixed inocula to ensure the variability of microorganisms. Both recommendations, standardized and mixed inoculum are contradictory and, although the variability of results depending on the test used is possibly a reflection of the behavior of microorganisms in natural ecosystems, the need for reproducibility and reliability in the data offered requires new research to have more robust biodegradability measurement methods.

In addition to the variability of existing biodegradability measurement methods and the problems associated with their use, Spanish water legislation does not establish limit values for the biodegradability of wastewater or reusable water. However, for other substances such as surfactants, the legislation even recommends which reference method should be used (EC 907/2006). Therefore, it is necessary to investigate in robust alternative methods that ensure the reproducibility of the results and allow to define a standardized protocol to evaluate the biodegradability of the effluents in urban and industrial treatment plants.

The present invention aims at the commercialization of the capsules containing the Pseudomonas putida bacteria as a standard inocula for the evaluation of "rapid biodegradability" avoiding the use of active sludge. Work prior to the patent study has been published demonstrating the feasibility of using said bacterium to measure the biodegradability of contaminated water although with significant differences with respect to the patent object kit. The authors of the invention developed a new method in 2008 to measure the biodegradability of contaminated water using liquid cultures of the P. putida bacteria and monitoring the biological elimination of Dissolved Organic Carbon (COD).

The method began to be used to evaluate the improvement of the biodegradability obtained by treating by means of photo-Fenton water contaminated with pesticides (MM Ballesteros Martín et al., Chemosphere 70, 1476-1483 (2008); MM Ballesteros Martín et al., 155 , 342-349 (2008)). Subsequently, its viability was demonstrated as a trial to assess biodegradability (A. Carc-Ripoll et al., Jounal of Hazardous Materials 162,1223-1227 (2009)).

To validate the results obtained with a single pesticide, and guarantee the generality of the trial, the same methodology was followed with a mixture of four commercial pesticides ((MM Ballesteros Martín et al., Water Research 43, 653-660 (2009)) and another of five commercial pesticides with a higher level of initial contamination ((MM Ballesteros Martín et al., Water Research 43.3838-3848 (2009)). The research carried out for the use of this method is developed in the Doctoral Thesis of Day. Me de la Menta Ballesteros Martín entitled "Elimination of non-biodegradable pesticides in waters by coupling solar photocatalysis and biological oxidation" defended at the University of Almeria in 2008.

In 2010, the authors of the invention published a comparative study that demonstrates the advantages of the trial compared to other alternative methods used to measure biodegradability ((MM Ballesteros Martín et al., Ecotoxicology and Environmental Safety 73, 1189-1195 (2010)) that aroused the interest of the scientific community in this new essay.

In order to extend this method to all those interested, it is proposed in this invention to standardize the procedure for its commercialization by means of lyophilized inocula capsules of the P. putida bacteria and an adequate proportion of mineral salts. To the same end, a new method of biodegradability assessment is presented by monitoring the Chemical Oxygen Demand.

Description of the invention

The "rapid biodegradability" measurement test using Pseudomonas putida bacteria is based on the use of capsules containing an adequate proportion of lyophilized bacteria and mineral salts that are added to the water whose biodegradability you want to evaluate.

First, the lyophilized bacteria is hydrated, mixing in a sterile tube the contents of the capsule a sterile 0.9% NaCl solution. In this way the standard inoculum is obtained which will be added later to the problem water.

A portion of inoculum is incubated next to the problem water in an Erlenmeyer flask that is placed in an orbital shaker at 150 rpm maintaining an incubation temperature of 30 ° C. A sterile aliquot is collected every 24 h and the COD is measured to a constant value (3-5 days).

Finally, biodegradation efficiency is calculated by the expression:

COD - (COD-COD) The = �100 COD�

where COD is the initial COD provided by the organic matter present in the water

contaminated, COD is the COD measured at the end of the test and COD is the COD

remnant obtained from a white test. If the biodegradation efficiency exceeds 40%, the contaminated compound or water can be considered to be "rapidly biodegradable."

Preferred Embodiment of the Invention

The bacterium Pseudomonas putida CECT 324 was obtained from the Spanish Type Culture Collection (Valencia, Spain). The cultures were maintained in Petri dishes for 24 hours at 30 ° C. with culture medium containing ox extract, 1 g L- \ yeast extract, 2 g L-1, peptone, 5 g L- \ NaCl, 5 g L -1 Y agar, 15 g L-1 at pH 7.2. Once the colonies were formed, one was collected and cultured in liquid medium in a 25 mL Erlenmeyer flask containing 5 mL of the previous medium (with the exception of agar). This solution was kept under stirring for 24 hours and distributed in sterile tubes to which 87% (v / v) glycerin solution was added in a 4/1 ratio (glycerin culture / solution). Once agitated, each tube was introduced in liquid nitrogen and stored at -70 ° C to obtain a bacterial stock.

Then, 200 t..tL of said stock was incubated in a 250 mL flask containing 50 mL of liquid medium (ox extract, 1 g L-1, yeast extract, 2 g L- \ peptone, 5 g L- 1 Y NaCl, 5 g L-1 at pH 7.2) for 24 h at 30 ° C. In order to obtain a high amount of biomass, larger volumes of flasks can be used following the same proportions. The biomass thus obtained was centrifuged for 15 min at 7000 rpm, washed with 0.9% sterile NaCl solution three times and the bacterial pellet was lyophilized for 24 h.

The lyophilized bacterium was encapsulated by adding 1 g of lyophilized bacteria and 5.5 g of NH4 C1, 5.5 g of K2HP04, 5.5 g of KH2P04, 5.5 g of MgS04�7H20, 0.11 in each capsule g of CaCl2�2H20, 0.11 g of MnS04�H20, 0.09 g of Fe (NH4) 2 (S04) 2�6H20, 0.02 g of ZnS04�7H20 and 0.02 g of nitrilotriacetic acid . The vehicle for encapsulation should preferably be insoluble in water and inert or in the form of a blister. With the contents of a capsule, the standard inoculum was prepared by mixing in a sterile tube the bacteria and mineral salts contained in the capsule and 10 mL of a sterile 0.9% NaCl solution. It was stirred until completely dissolved.

Then, the pH of the problem water was adjusted to 7, adding NaOH or H2S040.1 M and filtered through 0.80 J.1m filters, since if the filtration is done with smaller filters, part of Organic matter can be retained in it. A 50 mL aliquot was poured into a 250 mL Erlenmeyer flask (each test was performed in triplicate), 1.5 mL of standard inoculum was added and the flasks were capped with cotton plugs. Next, the flasks were placed on an orbital shaker at 150 rpm maintaining an incubation temperature of 30 ° C.

A 2 mL aliquot was collected every 24 h with a sterile pipette in a laminar flow cabinet and the OQO was measured to a constant value (3-5 days). The OQO can be measured by using spectrophotometric kits (A = 448 nm) suitable for the measuring range containing K2Cr20 7, H2S04, Ag2S04 and HgS04 as reagents and incubating at 148�C for 2 h. The method can be used for diluted samples taking into account that a large excess of OQO can lead to the indication of results outside the measurement range. In this case it is recommended to carry out a likelihood control of the measurement results by dilution. All measurements are made in duplicate and periodically a standard solution is measured.

Finally, biodegradation efficiency was calculated by the expression:

COD - (DQOr-COD) The = �100

. COD�

5 where COD is the initial COD provided by the organic matter present in the water

contaminated, COD is the COD measured at the end of the test and COD is the COD

remnant obtained from a white test. If the biodegradation efficiency exceeds 40%, the contaminated compound or water can be considered to be "rapidly biodegradable."

10 A blank test (in triplicate) was carried out in the same way as with the problem water but using distilled water. Likewise, a viability test of the inoculum was performed by inoculating for 24 h at 150 rpm and 30�C with 200 J.1L of the stock of polished P. flasks of 250 mL containing 50 mL of medium recommended by the

15 CECT for bacteria (ox extract, 1 g L- ', yeast extract, 2 g L- "peptone, 5 g L-" NaCl, 5 g L-' Y agar, 15 g L - ') at pH 7.2 And the optical density was measured at 600 nm. All materials were sterilized by autoclave at 126 ° C and 2 atm for 20 minutes before use.

Claims (2)

1. The invention consists of encapsulating an adequate proportion of bacteria P. lyophilized putida as a standard microorganism and a mixture of mineral salts. Each capsule contains one gram of lyophilized bacteria and 0.16 g of NH4C1, 0.16 g of K2HP04, 0.16 g of KH2P04, 0.16 g of MgS04� 7H20, 0.11 g of CaCl2�2H20, 0, 11 g of MnS04�H20, 0.08 g of Fe (NH4) 2 (S04) 2�6H20, 0.02 g of ZnS04�7H20 and 0.02 g of n-itrilotriacic acid. 2. The procedure to assess the rapid biodegradability of water through The method of protection consists in: a) Preparation of a P. putida inoculum by dissolving and homogenizing the contents of the capsule prepared according to claim 1 in 10 mL of a sterile 0.9% NaCl solution. b) Inoculation of 50 mL of the water under analysis (adjusted to pH 7 and sterilized by filtration) with 1.5 mL of the previously described inoculum, in an Erlenmeyer flask of 250 mL capacity at 30 ° C temperature and stirring Orbital c) Sampling and measurement of COD. Repeat every 24 hours for 3-5 days until a constant COD value is obtained (variation less than 10% between two consecutive days). d) Determination of rapid biodegradability by calculating the efficiency of biodegradation: COD - (COD-COD) The = �100 COD where DQD is the initial COD provided by the organic matter present in the contaminated water, COD I is the COD measured at the end of the test and COD is the remaining COD obtained from a white test.