Mixture of microorganisms for treating wastewater

Abstract

The present invention relates to a mixture of heterotrophic microorganisms comprising the bacterial strains Pedobacter sp., Pseudomonas sp. (extremaustralis) and Variovorax paradoxus. These microorganisms form a biofilm and are able to improve water quality and reduce odorous emissions and pathogenic agents in wastewater, preferably in liquid industrial waste (LIW). The present invention also discloses a device for storing and / or releasing the biofilm produced by the mixture of microorganisms, the use of the biofilm, the mixture or the device for reducing pollutants and volatile compounds in water treatment plants, lagoons, homogenisation wells, retention ponds, inter alia, of the agricultural sector.

Description

[0001] MIXTURE OF MICROORGANISMS FOR WASTEWATER TREATMENT

[0002] BRIEF DESCRIPTIVE MEMORANDUM DESCRIPTION OF THE INVENTION

[0003] The present invention describes a mixture of heterotrophic microorganisms comprising the bacterial strains Pedobacter sp. (registered with INTA under accession number RGM 3612), Pseudomonas sp. (extremaustralis) (registered with INIA under accession number 3613), and Variovorax paradoxus (registered with INIA under accession number RGM 3300), which are non-antagonistic and form a stable biofilm. The biofilm produced by this mixture of microorganisms has the capacity to improve water quality, reduce odor emissions, and decrease pathogens in wastewater, preferably in industrial liquid waste (ILW).

[0004] Additionally, the present invention discloses a device for storing and / or releasing the biofilm produced by the mixture of microorganisms, the use of the biofilm, the mixture or the device for the reduction of contaminants and volatile compounds in water treatment plants, lagoons, homogenization wells, and / or retention ponds, among others, in the agricultural sector.

[0005] It is worth noting that the mixture, the biofilm and the device of the present invention do not generate by-waste, unlike chemical and / or aromatic masking solutions that have the potential to generate additional costs for the post-treatment of chemical or organic waste, in addition to the fact that the degradation of contaminants allows the recirculation of water resources.

[0006] DESCRIPTION OF THE STATE OF THE ART

[0007] The presence of industrial liquid waste (ILW), which consists of organic waste in wastewater from industrial activity, including remains of various plant species, animal excrement, seeds, food scraps, plant waste, or mixtures thereof, has the capacity to ferment, particularly in intensive livestock, fishing, agriculture, and forestry operations. This generates a negative impact on the environment and on communities living near industrial areas. Some researchers estimate that animal agriculture accounts for 27% of total human water use worldwide for livestock production (including water used to grow feed grains). When water resource management is problematic, water quality can decline, and contamination can occur in livestock and poultry facilities with phosphates and nitrates, pathogens (viruses, bacteria, and protozoa), antibiotics, and androgenic agents.Furthermore, it has a significant environmental impact by releasing various gases and unpleasant odors. In particular, wastewater storage ponds pose a health problem for the communities surrounding any industrial site or facility that uses them, as they are accompanied by strong, foul odors that negatively affect the health of workers and make them extremely difficult to tolerate, along with the associated negative impacts. On the other hand, the limited availability of natural resources such as water is the primary reason for focusing on wastewater recovery.

[0008] In Chile, agriculture is concentrated between the Coquimbo and Los Lagos regions, covering most of the national territory, with 270,000 farmers working 30.45 million hectares, 40% of the continental land area. Our country has expressed its intention to increase the productivity of the agricultural sector to become an international food supplier. Considering that agricultural production has significant environmental impacts, affecting water, air, and soil, it is crucial to develop a sustainable agricultural model for the future, given that exports of natural resources are finite. Currently, water consumption is significant in agriculture (84.5%) and mining (6.5%).For this reason, the Government of Chile has declared that water is a priority resource and that scientific and technological strategies must be strengthened to optimize the use of water resources in industries that use this resource.

[0009] One way to address this is through the use of diverse wastewater treatment technologies, encompassing physical, chemical, and biological processes. Physical wastewater treatment involves the removal of contaminants without altering their biochemical properties and is generally carried out after chemical and biological treatments. Common physical techniques include screening, flotation, flow equalization, membrane-based technologies, and thermal treatments. On the other hand, chemical unit processes frequently used in wastewater treatment include precipitation, adsorption, disinfection, chlorination, neutralization, and chemical exchange, among others, all of which aim to improve water quality (Collivignarelli et al., 2018).

[0010] However, many of these treatments are costly, require high maintenance, and demand complex operational configurations (Singh D et al., 2022). Therefore, there is a need to replace these technologies with more sustainable and environmentally friendly alternatives that promote the use of microorganisms, as they have proven to be more effective in water treatment processes (Sharma M et al., 2023).

[0011] To solve the problems associated with wastewater, the present invention describes a mixture of heterotrophic microorganisms comprising the bacterial strains Pedobacter sp. (ENRIL-4, deposited with accession number RGM 3612 before INIA), Pseudomonas sp. (extremaustralis) (ENBIO 7, deposited with accession number 3613 before INIA) and Variovorax paradoxus (BIOPROC-C13, deposited with accession number RGM 3300 before INIA), which form a biofilm and have the ability to improve water quality, reduce odorous emissions and pathogens in industrial liquid waste (RILES).

[0012] Additionally, a device for storing and / or releasing the biofilm produced by the mixture of microorganisms is disclosed, as well as the use of the biofilm, the mixture or the device for the reduction of contaminants and volatile compounds in water treatment plants, lagoons, homogenization wells, retention ponds, among others, in the agricultural sector.

[0013] DESCRIPTION OF THE INVENTION

[0014] The present invention relates to a mixture of heterotrophic microorganisms comprising the bacterial strains Pedobacter sp. (ENRIL-4, registered with INIA under accession number RGM 3612), Pseudomonas sp. (extremaustralis) (ENBIO 7, registered with INIA under accession number 3613), and Variovorax paradoxus (BIOPROC-C13, registered with INIA under accession number RGM 3300). Preferably, these strains are able to tolerate a wide temperature range (18-37°C), high concentrations of ammonium (>200 mg / L) and sulfates (>100 mg / L), and possess hydrolytic enzymes, such as lipase, and exhibit hemolytic activity.

[0015] In an even more preferred modality, the mixture of these three strains forms a stable biofilm. The mixture and the biofilm have the capacity to improve water quality, reduce odor emissions, and decrease pathogens in industrial liquid waste (ILW). Preferably, the mixture of microorganisms and the biofilm it forms reduce contaminant levels according to the parameters of Chilean Standard NCh.1.333 Of78 in ILW from the agricultural industry.

[0016] On the other hand, the present invention describes a device for storing and releasing the biofilm produced by the mixture of microorganisms.

[0017] In another embodiment, the present invention relates to the mixture of microorganisms in solid form obtained by spray drying or freeze-drying techniques.

[0018] Furthermore, the present invention discloses the use of the biofilm, mixture, or device for reducing pollutants and volatile compounds in wastewater treatment plants, including those for water, lagoons, homogenization wells, retention ponds, and other facilities in the agricultural sector. In the context of this invention, the agricultural sector refers to, but is not limited to, the livestock, fishing, intensive timber, and wine industries, as well as agriculture.

[0019] Also described is a method for treating wastewater comprising the application of the mixture of microorganisms, the biofilm or a device comprising one of these in the water effluent.

[0020] In one application method, the mixture, biofilm, or device is applied at least every two months, preferably every three months. This interval between applications is longer compared to bacterial powder-based treatments applied monthly and aromatic or chemical treatments applied weekly.

[0021] It is important to highlight that the mixture, the biofilm, and the device do not generate by-products, unlike chemical and / or aromatic masking solutions that have the potential to generate additional costs due to the post-treatment of chemical or organic waste. Furthermore, the degradation of contaminants allows for the recirculation of water resources. DESCRIPTION OF THE FIGURES

[0022] Figure 1: Bacterial strain Variovorax paradoxus (BIOPROC-C13 or RGM 3300) deposited in the Chilean Collection of Microbial Genetic Resources – INIA, at a temperature of 25 °C and 48h of incubation.

[0023] Figure 2: Bacterial strain Pedobacter sp. (ENRIL-4) on 1% TSA agar 200 mg / L NH4+ at a temperature of 25 °C and 48 h of incubation.

[0024] Figure 3: Bacterial strain Pseudomonas sp, (extremaustralis) (ENBIO 7) on 1% TSA agar 200 mg / L NH4+ at a temperature of 25 °C and 48 h of incubation.

[0025] Figure 4: Microscopic images (stained with fluorochromes) of polyethylene structures colonized by bacterial biofilm formed in 120L reactors. Live bacteria are shown in green and dead bacteria in red. (A) 10X magnification (B) 25X magnification (C) 40X magnification (D) 63X magnification.

[0026] Figure 5: Micrographs of membranes colonized by bacteria forming biofilms. A. Membrane with biofilms (1 mm) observed with a scanning electron microscope, B. Micrograph of a membrane with biofilms (40X) obtained with a confocal laser microscope. Live bacteria are visualized in green and dead bacteria in red, using Syto 9 fluorochromes and propidium iodide (IP) for staining.

[0027] Figure 6: Prototype installed in Viña Aresti.

[0028] Figure 7: Graphic summary, percentage decrease of analytes measured in pilot RILES pool.

[0029] Figure 8: Parameters measured in pilot RILES pool versus time.

[0030] Figure 9: Micrographs of biofilms on membranes. A, Membrane with bacterial biofilm observed with a scanning electron microscope at time 0 days in a pilot pool. B. Membrane with bacterial biofilm observed with a scanning electron microscope at time 60 days in a pilot pool.

[0031] EXAMPLES

[0032] Example 1: Obtaining samples and selecting bacterial strains Obtaining samples and culturing strains

[0033] A bacterial collection was obtained from the effluent of the industrial wastewater treatment plant (RILES) at Viña Aresti SA, located at Fundo Bellavista s / n, Río Claro, Molina, Chile. This source was selected to obtain isolates capable of metabolizing high concentrations of nitrogen and sulfur compounds.

[0034] Specifically, sampling points were selected at Viña Aresti S.A. to collect samples of both wastewater (effluent and influent) and biofilms from the winery's effluent system. For wastewater samples, 100 mL were collected in sterile bottles (in triplicate), and for biofilm collection, a sterile swab was used to swab an area of ​​approximately 100 cm². 2Samples were collected from the surface of the influent system and stored in PBS buffer in a 15 mL Falcon tube (in triplicate). All samples were stored in a temperature-controlled cooler at 4°C for transport to the laboratory and subsequently stored at this same temperature for processing (Sánchez-Romero et al., 2019). From the collected samples, 100 µL aliquots were inoculated onto different culture media to count various microorganisms. The media used were: R2 Agar (total bacterial count), PDA Agar (total fungal count), and King B Agar (partial count of Pseudomonas sp.). Isolated colonies were stored in a culture collection at -20°C.

[0035] Using the same samples, the aim was to enhance the presence of heterotrophic bacteria capable of metabolizing ammonium in environments with organic matter (Nyerges et al., 2010; Ma et al., 2022). For this purpose, a medium was prepared with a concentration of 200 mg / L of ammonium (ammonium sulfate) and 1% tryptic soy broth in a 1-liter amber Schott bottle; agar plates were also prepared with this same composition. Samples from wastewater were enriched separately from those from biofilms for a period of 3 weeks at 25°C under constant agitation at 100 rpm.

[0036] Isolation of strains and extraction of bacterial DNA

[0037] After three weeks of incubation in the enrichment medium, serial dilutions were performed for both the enrichment of wastewater and biofilms to perform a total bacterial count and subsequently isolate strains. These dilutions were then plated onto plates prepared from the enrichment broth (200 mg / L ammonium and 1% tryptic soy broth). The plates were incubated at 25°C for 72 hours. Strains were selected based on their distinctive morphology and by considering those that were predominantly present at higher concentrations. This was done by streaking individual colonies with a loop. This procedure was repeated three times to ensure a single colony and minimize contamination by other strains.

[0038] Once the strains were isolated, each was grown in a culture medium containing 200 mg / L ammonium and 10% tryptic soy broth and incubated for 2 days at 25°C. DNA was extracted from bacterial pellets obtained by centrifugation at 8000 rpm using the commercial EZN A® Bacterial DNA Kit. To confirm the integrity of the extracted DNA, a standardized molecular weight marker (100 to 10,000 base pairs) was used (1Kb DNA Ladder, Promega). The DNA was then visualized using a UV transilluminator (UVITEC Cambridge).

[0039] Molecular characterization of the strains

[0040] For molecular characterization of the isolated strains, the 16S rRNA fragment was amplified using the 27F and 1492R primers specific to this region in bacteria (Zoetendal et al., 2001; Rosseli et al., 2016; Straub et al., 2020) by endpoint PCR (Polymerase Chain Reaction) and subsequently sequenced. DNA extracts from the samples were sent to the AUSTRAL-omics Genomic Laboratory at the Austral University of Chile for gene amplification and subsequent sequencing using an ABI3500 System sequencer (Applied Biosystems™). Once the data were obtained, the raw sequences were analyzed using 4Peaks® software to select those with good quality and integrity. The Basic Local Alignment Search Tool (BLAST) platform (National Center for Biotechnology Information, USA) was used to align the sequences with the existing database.The genus and / or species of each was determined using the highest percentage of identity, also considering the percentage of gene coverage (Query cover) and the lowest randomness probability value (E-value).

[0041] Selection of bacterial strains

[0042] A screening methodology was used to select the bacterial strains, evaluating their capacity to metabolize nitrogenous (ammonium > 200 mg / L) and sulfurous (sulfides > 100 mg / L) compounds. Ammonium and sulfide consumption was measured in triplicate using spectrophotometry with the Spectroquant® ammonium kits (Merck), based on EPA standards 354.1 and 350.1, and the S sulfide kit. z “ Spectroquant® (Merck), respectively.

[0043] In addition, the ability of the strains to tolerate a wide range of temperatures (18-37°C) was evaluated by incubating them at a concentration of 10 5CFU / mL were measured in triplicate for 48 hours. Subsequently, microorganisms were counted using the microdrop method.

[0044] The presence of enzymes with lipolytic and hemolytic activity was also evaluated. Hemolytic activity was determined following the procedure described by Kavitha, M., et al. (2018), with modifications. Strains with hemolytic activity were discarded. For the detection of lipolytic activity, the strains were plated on agar plates composed of Tween 80 (4 ml / L), calcium chloride (1 g / L), tryptone (15 g / L), soy peptone (5 g / L), sodium chloride (5 g / L), and agar (15 g / L). The plates were incubated at 18°C ​​for 48 hours, after which areas of turbidity indicating lipase activity were observed.

[0045] Finally, a literature search was conducted to verify that the strains were not phylogenetically related to pathogenic species, ruling out their pathogenicity. From all the strains obtained, three bacterial strains were selected: Variovorax paradoxus (deposited with accession number RGM 3300 at INIA and henceforth identified only as “RGM 3300”) (see Figure 1), Pedobacter sp. (ENRIL-4) (see Figure 2), and Pseudomonas sp. (extremaustralis) (ENBIO 7) (see Figure 3). These strains have 16S rRNA fragment nucleotide sequences with SEQ ID NOs: 1, 2, and 3, respectively.

[0046] Table 1 below shows the results obtained from the previously mentioned trials to select the strains, as well as the ammonia resistance of the mixture of the three strains as detailed in Example 2 below:

[0047] Table 1: Attributes of the selected strains and the mixture Activity Growth to Consumption

[0048] different T (°C)

[0049] Bacterial strain Hemolytic Lipase 25 32 37 Ammonium (%) Ammonium (%) Variovorax mixture -h - 4 -h 4 50

[0050] paradoxus (RGM

[0051] 3300)

[0052] Pedobacter sp. - 4- 4 4 70 58.12

[0053] (ENRIL-4)

[0054] Pseudomonas sp. 4- 4- 4- 50

[0055] (extremaustalis)

[0056] (ENBIO-7)

[0057]

[0058] Example 2: Culturing the strains together and analyzing the biofilm they form

[0059]

[0060] The strains Pedobacter sp. (ENRIL-4), Pseudomonas sp. (extremaustralis) (ENBIO 7), and Variovorax paradoxus (RGM 3300) were cultured in 120 L bioreactors with a polymeric surface (70% of the total reactor volume), using 200 mg / L ammonium sulfate and 1% tryptic soy broth as the culture medium. The culture was carried out for 5 days with constant aeration (9 mg / L oxygen) and a temperature between 15–21 °C. The effectiveness of the culture in generating a mixture was verified using electron micrographs. A two-photon spectral confocal laser microscope (LSM780 NLO Zeiss) was used, and the images were analyzed using ZEN 2011 SP3 (black edition) software (see Figure 4). Bacterial viability tests were performed using Live / Dead staining (BacLight Viability Kit Invitrogen, USA) according to the manufacturer's instructions.

[0061] The biofilm formed homogeneously on the surface of the polymeric structure, with 80% live bacteria.

[0062] These tests demonstrate that the 3 selected strains do not exhibit antagonism between them, so they are able to coexist and form biofilms effectively.

[0063] In addition, ammonium consumption was analyzed using spectrophotometry with the Spectroquant ammonium kit (Merck) (the technique was based on EPA 354.1 and 350.1). Analyses were performed on each strain individually and on strains mixed at a concentration of 10 6 With an initial ammonia concentration of 200 mg / L and a UFC / mL concentration of 200 mg / L, consumption was about 50% in all treatments at a time of 96h (see Table 1 above).

[0064] Example 3: Treatment of liquid wine waste Microorganisms were grown on high porosity membranes (1 m 3, 30 ppi) in batch reactors, at a constant temperature of 20°C and a dissolved oxygen of 9 mg / L, for a period of 72 hours.

[0065] At Viña Aresti, a pilot wastewater treatment system using biological membranes was installed (see Figures 5A and 5B). This modular system has a treatment capacity of 33.54 m³ 3 It consists of a double-layered PVC pool, reinforced with an intermediate polyester mesh for greater abrasion resistance. The electromechanical system includes a self-priming electric pump, an electrical panel, and fluid transport lines, which allow for the extraction of industrial liquid waste (ILW) from the main Viña Aresti pool to the pilot pool. In addition, a perimeter fence was implemented to protect local wildlife (see Figure 6).

[0066] The prototype was implemented and the physicochemical parameters were measured during April and May, coinciding with the grape harvest, when the highest concentration of organic waste is generated in the plant. Samples were taken every 15 days, and the corresponding analyses were performed at the accredited Biodiversa laboratory, located in the Maule Region, Chile (see Table 2, Figures 7-8).

[0067] Table 2: Analysis of pilot RILES pool performed by accredited external laboratory Biodiversa - Maule

[0068] Decrease in Parameters Day 1 Day 15 Day 30 Day 60 Percentage (%) Coliforms 2.3 1.3 1.3

[0069] 3.0 E+03 99.5 (NMP / 1000) E+01 E-í-01

[0070] Biochemical Demand

[0071] Oxygen (BOD) 8,472.75 4,837.00 4,857.47 3,964.1 52.21 (mg / L)

[0072] Chemical Demand of

[0073] 17,350.00 9,720 9,970.00 6970 59.83 Oxygen (COD) (mg / L)

[0074] Dissolved sulfate (mg / L) 253.1 198.7 254 97.9 61.3 Nitrate nitrogen

[0075] 3.18 2.14 2.46 0.4 87.4 (mg / L)

[0076] Total Dissolved Solids 6,806.00 2,814.00 2,970.00 2,102.00 69.11 (mg / L)

[0077]

[0078] In Figure 9A, at least two different bacterial morphologies can be observed adhering to the polyethylene mesh, including two distinct sizes of bacilli. In Figure 9B, at least three bacterial morphologies are identified, notably bacillary and coccoid forms. It is important to note that, after 60 days of exposure of the mesh to the industrial wastewater, bacterial colonization persists, maintaining the initial inoculation morphology on the membrane. This suggests that the biofilm remains stable for 60 days under the tested conditions.

[0079] Conclusions

[0080] During the 60-day biological treatment experiment in the winery wastewater (WWW), a significant reduction in BOD and COD was observed in the pilot tank, achieving a decrease of over 50%, along with improvements in the other measured parameters. The treatment proved effective under the tested conditions, showing a notable percentage reduction in the evaluated parameters after 15 days, without variations in pH, temperature, or color of the treated WWW. Furthermore, the bacterial morphology of the inoculum persisted over time, suggesting that the biofilm adapts to the environmental conditions of the winery WWW.

[0081] REFERENCES

[0082] Collivignarelli MC, Abbá A, Benigna I, et al. Overview of the main disinfection 890 processes for wastewater and drinking water treatment plants. Sustainability.

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[0084] Kavitha, M., Raja, M. & Perumal, P. Evaluation of probiotic potential of Bacillus spp. isolated from the digestive tract of freshwater fish Labeo calbasu (Hamilton, 1822). Aquaculture Reports 11, 59-69, doi:https: / / doi.org / 10.1016 / j.aqrep.2018.07.001 (2018). Nyerges, G., Han, S. K., & Stein, L. Y. (2010). Effects of ammonium and nitrite on growth and competitive fitness of cultivated methanotrophic bacteria. Applied and environmental microbiology, 76(16), 5648-5651. https: / / doi.org / 10.1128 / AEM.00747-10

[0085] Ma, S., Huang, S., Tian, Y., & Lu, X. (2022). Heterotrophic ammonium assimilation: An important driving force for aerobic denitrification of Rhodococcus erythropolis strain Y10. Chemosphere, 29I(Pt 3), 132910. https: / / doi.org / 10.1016 / j.chemosphere.2021.132910

[0086] Sánchez-Romero, MI, Garcia-Lechuz Moya, JM, González López, JL, & Orta Mira, N. (2019). Collection, transport and general processing of clinical specimens in a microbiology laboratory. Recogida, transporte y proceso general de las muestras en el laboratorio de Microbiología. Enfermedades intenciones y microbiología clínica (English ed.), 37(2), 127-134. https: / / doi.org / 10.1016 / j.eimc.2017.12.002.

[0087] Singh D, Goswami RK, Agrawal K, et al. Bioinspired remediation of wastewater: a contemporary approach for environmental clean up. Curr Res Green Sustain Chem.

[0088] 2022;5:1-165.

[0089] Sharma M, Agarwal S, Agarwal Malik R, Kumar G, Pal DB, Mandal M, Sarkar A, Bantun F, Haque S, Singh P, Srivastava N, Gupta VK. Recent advances in microbial engineering approaches for wastewater treatment: a review. Bioengineered. 2023 Dec;14(l):2184518. doi: 10.1080 / 21655979.2023.2184518. PMID: 37498651; PMCID: PMC10376923.

Claims

CLAIMS 1. Mixture of microorganisms, CHARACTERIZED in that it comprises the bacterial strains Pedobacter sp., Pseudomonas sp. (extremaustralis) and Variovorax paradoxus.

2. The mixture of microorganisms according to claim 1, CHARACTERIZED in that the bacterial strains are Pedobacter sp. (deposited with accession number RGM 3612 before INIA), Pseudomonas sp. (extremaustralis) (deposited with accession number 3613 before INIA) and Variovorax paradoxus (deposited with accession number RGM 3300 before INIA).

3. The mixture of microorganisms according to claim 1 or 2, CHARACTERIZED in that they form a stable biofilm.

4. The mixture of microorganisms according to any of claims 1 to 3, CHARACTERIZED in that it is in solid form obtained by spray drying or freeze-drying techniques.

5. Storage and / or release device, CHARACTERIZED in that it comprises the mixture of microorganisms according to any of claims 1 to 4 or the biofilm of said mixture of microorganisms.

6. Use of the mixture of microorganisms according to any of claims 1 to 4, of the biofilm according to any of claims 1 to 4, or the storage and / or release device according to claim 5, CHARACTERIZED in that it serves for the reduction of contaminants and volatile compounds in wastewater.

7. The use according to claim 6, CHARACTERIZED in that the waste liquids correspond to water, lagoons, homogenization wells, and / or retention ponds.

8. The use according to claim 6 or 7, CHARACTERIZED in that the wastewater corresponds to industrial waste (RILES) from the agricultural industry.

9. A method for treating wastewater, CHARACTERIZED in that it comprises the application of the mixture of microorganisms according to any of claims 1 to 4, the biofilm according to any of claims 1 to 4, or the storage and / or release device according to claim 5, in the wastewater effluent.

10. The method according to claim 9, CHARACTERIZED in that it optionally comprises applying the mixture, the biofilm or the device at a frequency of at least two months.

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