[go: up one dir, main page]

WO2024237867A1 - Utilisation de souches bactériennes résistantes à l'antimoine pour la bioremédiation et procédé de bioremédiation utilisant ces mêmes souches bactériennes - Google Patents

Utilisation de souches bactériennes résistantes à l'antimoine pour la bioremédiation et procédé de bioremédiation utilisant ces mêmes souches bactériennes Download PDF

Info

Publication number
WO2024237867A1
WO2024237867A1 PCT/SK2023/050010 SK2023050010W WO2024237867A1 WO 2024237867 A1 WO2024237867 A1 WO 2024237867A1 SK 2023050010 W SK2023050010 W SK 2023050010W WO 2024237867 A1 WO2024237867 A1 WO 2024237867A1
Authority
WO
WIPO (PCT)
Prior art keywords
aeromonas
antimony
bacterial strains
strains
bioremediation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/SK2023/050010
Other languages
English (en)
Inventor
Hana MAJEROVA
Ivona KAUTMANOVA
Zuzana KONYARIKOVA
Dana SZABOOVA
Bronislava VOLEKOVA
Jan KAUTMAN
Christian PUHR
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Slovak National Museum Natural History Museum
Original Assignee
Slovak National Museum Natural History Museum
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Slovak National Museum Natural History Museum filed Critical Slovak National Museum Natural History Museum
Priority to PCT/SK2023/050010 priority Critical patent/WO2024237867A1/fr
Publication of WO2024237867A1 publication Critical patent/WO2024237867A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/341—Consortia of bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C02F2101/20—Heavy metals or heavy metal compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • HFO hydrous ferric oxides
  • Antimony is a potentially toxic element occurring naturally in many foods, but it may cause significant damage to human health at higher concentrations. It has negative health effects on human organs such as liver and kidneys, may damage fertility or the unborn child, is suspected of causing cancer, may cause harm to breast-fed children, may cause damage to organs through prolonged or repeated exposure and is harmful to aquatic life with long lasting effects.
  • antimony In contaminated areas, it enters the food chain either directly, via its solubility in water, or through plants, fungi and bacteria and their subsequent consumption by animals (invertebrates and vertebrates). Therefore, antimony must be considered a risk factor that seriously endangers the functionality and health of ecosystems in the long-term view.
  • HFOs include several minerals, such as ferrihydrite, akaganeite, feroxyhyte, goethite, lepidocrocite and limonite. They are poorly crystalline, highly porous, have large surface areas and, thanks to this, are excellent sorbents of various potentially toxic elements. Sorptive properties of HFOs depend on the pH values of the surrounding water, the chemical composition of the water, and the ratio of the amount of dissolved trace metals to the amount of hydrous iron oxides.
  • HFOs harbor a remarkable ability to sequester Sb from the environment by adsorbing antimonate (Sb(V)) and antimonite (Sb(III)) ions on its surface (in some cases even into the structure) under the neutral and low acidic conditions, which is the case of most groundwater and soil water conditions.
  • Sb(V) antimonate
  • Sb(III) antimonite
  • the redox state of metals in HFO complexes, and Sb adsorption to HFO minerals are not stable, but they are constantly changing, either as the result of diverse geochemical interactions or as result of interactions with diverse living organisms, mainly bacteria, algae, and fungi.
  • Microorganisms inhabiting iron ochres are well adapted to this extreme environment and many of them, as we have concluded previously, are metabolically active over metals and metalloids accumulation, sorption and redox state maintenance.
  • the aim of this invention is to isolate pure cultures of bacterial strains, evaluate their individual metabolic potentials and testing their bioremediation potential, meaning mainly their ability to adsorb Sb on their surfaces, incorporate or accumulate the Sb within their cells, and their ability to metabolically contribute to chemical fixation and/or sorption of Sb in diverse compounds, i.e. by changing Sb redox state.
  • the aim of this invention is to provide bacterial strains for bioremediation use in polluted environment.
  • KSb(OH)e potassium hexa hydroxyantimonate
  • HFO hydrous ferric oxides
  • CFU colony forming units RNA - ribonucleic acid
  • BLASTn nucleotide Basic Local Alignment Search Tool NCBI - National Center for Biotechnology Information
  • ICP-MS inductively coupled plasma mass spectrometry PCR - polymerase chain reaction
  • the subject matter of this invention is 7 bacterial strains isolated form metal- and metalloid-polluted iron ochres from publicly approachable site nearby abandoned mine Buducnost (Pezinok, Western Slovakia).
  • the bacterial strains were characterized by sequencing their VI to V9 16S rRNA genomic regions (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7) and identified using BLASTn searches against 16S ribosomal RNA sequences (Bacteria and Archaea) at NCBI (https://blast.ncbi.nlm.nih.gov/).
  • the bacterial strains were characterized by estimating their growth performances on complex solid and liquid TS media and by characterizing them in terms of basic morphological features, i.e. texture, color and surface.
  • the isolated strains are stored at -80°C as glycerol stocks and grown and maintained on TS and TSA media at wide range of tested temperatures (4°C - 30°C).
  • the site from which the bacteria were isolated is known to harbor high average, although fluctuating, concentrations of metals and metalloids, and thus, the microorganisms, including bacteria, living at this site are likely to show certain degree of tolerance to increased concentrations of these metals and metalloids.
  • the average values of the measured concentrations of concerned metals and metalloids are listed in Tab. 1.
  • the first aspect of this invention is the use at least one bacterial strain selected from a group consisting of Aeromonas sp. A21 (16S rRNA genomic regions SEQ ID NO: 1), Aeromonas sp. A13 (16S rRNA genomic regions SEQ ID NO: 2), Aeromonas sp. A60A (16S rRNA genomic regions SEQ ID NO: 3), Acinetobacter sp. A14 (16S rRNA genomic regions SEQ ID NO: 4), Buttiauxella sp. A58 (16S rRNA genomic regions SEQ ID NO: 5), Shewanella sp. A20A (16S rRNA genomic regions SEQ ID NO: 6) and Yersinia sp. A68 (16S rRNA genomic regions SEQ ID NO: 7) as natural hyperaccumulating strains for bioremediation of Sb from soil, water and iron ochres.
  • the second aspect of this invention is a method of bioremediation polluted soil, water, iron ochres and environment using at least one bacterial strain selected from a group consisting of Aeromonas sp. A21, Aeromonas sp. A13, Aeromonas sp. A60A, Acinetobacter sp. A14, Buttiauxella sp. A58, Shewanella sp. A20A and Yersinia sp.
  • A68 wherein said method comprises of steps: a) Application at least one of bacterial strain to the environment to be remediated, and b) Hyperaccumulation of antimony and other metalloids within bacterial biomass and/or increasing or decreasing binding affinities to other compounds and/or influencing the redox states of metals and metalloids in polluted environment and/or influencing their sorptive behavior.
  • the invention is collection of 7 stable bacterial cultures of isolated strains - Aeromonas sp. A21, Aeromonas sp. A13, Aeromonas sp. A60A, Acinetobacter sp. A14, Buttiauxella sp. A58, Shewanella sp. A20A and Yersinia sp. A68.
  • Each one of these strains may be applicable in unlimited biotechnological procedures requiring the aforementioned strains under the elevated concentrations of concerned metals and metalloids (marked with asterisk in Tab. 1), whereas the "elevated" means above limited concentrations in water and exceeding ID limits for soils according to the Slovak legislative.
  • the isolated bacteria may be used, for example, to influence the redox states of metals and metalloids in polluted environment, e.g. water, soil, iron ochres; to enhance or reduce their binding affinities to other compounds; to influence their sorptive behavior; to directly accumulate the metals and metalloids from soil, water, iron ochres and other natural and waste mixtures within their biomass; or to precipitate metal and metalloids compounds from natural and waste materials and their mixtures.
  • polluted environment e.g. water, soil, iron ochres
  • the isolated bacteria may be used, for example, to influence the redox states of metals and metalloids in polluted environment, e.g. water, soil, iron ochres; to enhance or reduce their binding affinities to other compounds; to influence their sorptive behavior; to directly accumulate the metals and metalloids from soil, water, iron ochres and other natural and waste mixtures within their biomass; or to precipitate metal and metalloids compounds from natural and waste materials
  • the inventors have determined that these strains grow in TS and TSA media supplemented with elevated concentrations of Sb (Tab. 2, Fig. 1 - Fig. 9) and accumulate Sb within their biomass with varying level of efficiency when cultivated in TS medium supplemented with 300 mg/l of Sb in form of KSb(OH)6 compared to the same strains cultivated in TS medium without Sb (Tab. 3).
  • strains Aeromonas sp. A21, Aeromonas sp. A13, Aeromonas sp. A60A, Acinetobacter sp. A14, Buttiauxella sp. A58, Shewanella sp. A20A and Yersinia sp. A68 can accumulate Sb within their biomass with higher efficiency than the control Pseudomonas strains A5 and A6 (isolated from the same site) and they assume that these strains create collection of Sb hyperaccumulating strains (see Tab. 2 and Tab. 3).
  • Sb hyperaccumulating strains here means that they can accumulate Sb within their biomass at least 5 times more efficiently than the control Pseudomonas A5 and A6 strains (fold increase in Tab. 3).
  • Each one of 7 Sb hyperaccumulating isolated strains may be applicable in any biotechnological procedure as described above where the accumulation, or possibly also precipitation, of Sb from the natural or waste materials and their mixtures is desirable.
  • Fig. 1 to Fig. 9 show representative growth curves of the bacterial strains, considered to be Sb resistant.
  • the strains were grown in liquid TS media either not supplemented (control) or supplemented with increasing concentrations of Sb.
  • the concentrations of Sb were measured post experiment and averaged, with the SD error rate 37.1 mg/l Sb at 300 mg/l of Sb.
  • TSA medium 1.5 % peptone from casein [VWR Chemicals], 0.5 % peptone from soy [VWR Chemicals], 0.5 % NaCI [Slavus], 1.5 % agar
  • liquid TS medium 1.5 % peptone from casein, 0.5 % peptone from soy, 0.5 % NaCI,
  • the media were prepared either, with standard distilled water or when appropriate the sterile filtered water collected at the sampling site was added instead.
  • TS media was mixed with sterile 50 % glycerol (1:1) and stored at -80 °C.
  • KSb(OH)6 SigmaAldrich
  • TS media a stock solution containing 4 g/l of Sb in TS media was prepared and autoclaved, then, after 1 day at room temperature, the undesired crystals were filtered out and the resulting medium was used in the experiments as the medium with the highest concentration of Sb.
  • the other liquid media for growth assays and Sb removal assays were prepared from this filtered stock by dilutions and the actual Sb concentrations were determined by ICP-MS.
  • the samples for isolation of bacterial strains were transported and stored at 4 °C. Before the isolation the samples which consisted of river water and ochre-sediment were vortexed and several dilutions were made. 100 pl of the dilutions were plated on TSA. Part of the media was made with river water and sludge to see if traces of antimony might influence the bacterial composition. Plates were incubated at 20, 25 and 30 °C for 3 days. Each day single cultures appearing were selected and passed on new TSA plates. The selection was performed due to differences in morphological features.
  • 515F (GTGCCAGCMGCCGCGGTAA, SEQ ID NO: 10)/1492R (CGGTTACCTTGTTACGACTT, SEQ ID NO: 11).
  • the PCR conditions were as follows: Initial denaturation for 5 min at 95 °C followed by 30 cycles of denaturation for 30 sec at 95 °C, annelation for 30 sec at 50 °C (for 27F/805R) or 30 sec at 49 °C (for 515F/1492R), elongation for 45 sec (for 27F/805R) or 1 min (for 515F/1492R) at 68 °C, ended by final elongation for 10 min at 68 °C. Reactions were run using the Hot Start Taq 2 Master Mix [NEB].
  • the same primers were used for sequencing.
  • the resulting sequences were combined into one sequence encompassing variable domains VI to V9 of the 16S rRNA, aligned and clipped at the 5' and 3' ends, where needed.
  • the redundant sequences were grouped together and representative sequence for each group was BLASTn classified against 16S ribosomal RNA sequences (Bacteria and Archaea) at NCBI (https://blast.ncbi.nlm.nih.gov/), see attached sequence listing part of the description.
  • TS + Sb represents the experiment where the Sb was added TS to the final concentration of 300 mg/l ⁇ 37.1 mg/l SD
  • fold increase represents the calculated fold increase in Sb concentration in TS + Sb media over the control TS media
  • CFU represents percentage of surviving cells in TS +Sb media compared to control TS media at the end of experiment.
  • This invention is industrially applicable in removing pollution namely Sb from the environment, for example in bioremediation.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Environmental & Geological Engineering (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Hydrology & Water Resources (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Water Supply & Treatment (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Soil Sciences (AREA)
  • Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne le processus de maintien de l'état d'oxydoréduction et/ou de biosorption de l'élément potentiellement toxique antimoine (Sb) par diverses souches bactériennes, isolées à partir d'ocres de fer riches en oxydes ferriques hydratés (HFO) présents dans les eaux s'écoulant des bassins de décantation sur le site d'une mine abandonnée. L'invention définit les souches bactériennes Aeromonas sp. A21, Aeromonas sp. A13, Aeromonas sp. A60A, Acinetobacter sp. A14, Buttiauxella sp. A58, Shewanella sp. A20A et Yersinia sp. A68 comme aptes à se développer dans un environnement d'ocres de fer pollué par des métaux et des métalloïdes et définit les conditions requises pour maintenir ces souches en laboratoire. L'invention concerne la capacité de ces bactéries à se développer en présence de concentrations élevées d'antimoine et également leur capacité à extraire l'antimoine du milieu de culture. Les souches bactériennes peuvent être utilisées pour la biorestauration de l'antimoine dans un environnement contaminé et, éventuellement, pour la bioproduction de Sb à partir de gisements de minerais ou de réservoirs naturels tels que les ocres de fer.
PCT/SK2023/050010 2023-05-17 2023-05-17 Utilisation de souches bactériennes résistantes à l'antimoine pour la bioremédiation et procédé de bioremédiation utilisant ces mêmes souches bactériennes Pending WO2024237867A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/SK2023/050010 WO2024237867A1 (fr) 2023-05-17 2023-05-17 Utilisation de souches bactériennes résistantes à l'antimoine pour la bioremédiation et procédé de bioremédiation utilisant ces mêmes souches bactériennes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/SK2023/050010 WO2024237867A1 (fr) 2023-05-17 2023-05-17 Utilisation de souches bactériennes résistantes à l'antimoine pour la bioremédiation et procédé de bioremédiation utilisant ces mêmes souches bactériennes

Publications (1)

Publication Number Publication Date
WO2024237867A1 true WO2024237867A1 (fr) 2024-11-21

Family

ID=86692679

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SK2023/050010 Pending WO2024237867A1 (fr) 2023-05-17 2023-05-17 Utilisation de souches bactériennes résistantes à l'antimoine pour la bioremédiation et procédé de bioremédiation utilisant ces mêmes souches bactériennes

Country Status (1)

Country Link
WO (1) WO2024237867A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003011487A1 (fr) * 2001-07-27 2003-02-13 Global Biosciences, Inc. Biorestauration de contaminants metalliques avec des bacteries utilisant des hydrocarbures
US6753179B2 (en) * 1999-12-08 2004-06-22 Allmighty Co., Ltd. Method for purification treatment of environmental pollutants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6753179B2 (en) * 1999-12-08 2004-06-22 Allmighty Co., Ltd. Method for purification treatment of environmental pollutants
WO2003011487A1 (fr) * 2001-07-27 2003-02-13 Global Biosciences, Inc. Biorestauration de contaminants metalliques avec des bacteries utilisant des hydrocarbures

Similar Documents

Publication Publication Date Title
Chen et al. Biostimulation of carbonate precipitation process in soil for copper immobilization
Li et al. Anaerobic bacterial immobilization and removal of toxic Sb (III) coupled with Fe (II)/Sb (III) oxidation and denitrification
Sedlakova-Kadukova et al. Bioaccumulation and biosorption of zinc by a novel Streptomyces K11 strain isolated from highly alkaline aluminium brown mud disposal site
Martins et al. Characterization and activity studies of highly heavy metal resistant sulphate-reducing bacteria to be used in acid mine drainage decontamination
Yang et al. Archaeal and bacterial communities in acid mine drainage from metal-rich abandoned tailing ponds, Tongling, China
Banerjee et al. Insight into Cr6+ reduction efficiency of Rhodococcus erythropolis isolated from coalmine waste water
Li et al. Anaerobic nitrate reduction with oxidation of Fe (II) by Citrobacter Freundii strain PXL1–a potential candidate for simultaneous removal of As and nitrate from groundwater
Garcia-Dominguez et al. Novel autotrophic arsenite-oxidizing bacteria isolated from soil and sediments
Park et al. Characterization of iron and manganese minerals and their associated microbiota in different mine sites to reveal the potential interactions of microbiota with mineral formation
Brantner et al. Depth-dependent geochemical and microbiological gradients in Fe (III) deposits resulting from coal mine-derived acid mine drainage
Gu et al. Sb (III)-resistance mechanisms of a novel bacterium from non-ferrous metal tailings
Shao et al. Autotrophic denitrification and its effect on metal speciation during marine sediment remediation
Said et al. Using Sarcocornia fruticosa and Saccharomyces cerevisiae to remediate metal contaminated sediments of the Ria Formosa lagoon (SE Portugal)
Wang et al. Fabricating biogenic Fe (III) flocs from municipal sewage sludge using NAFO processes: Characterization and arsenic removal ability
Mao et al. Removal of manganese in acidic solutions utilizing Achromobacter sp. strain QBM-4 isolated from mine drainage
Panneerselvam et al. Potential of novel bacterial consortium for the remediation of chromium contamination
Sorour et al. Microbe-plant interaction as a sustainable tool for mopping up heavy metal contaminated sites
Zhu et al. Stable immobilization of uranium in iron containing environments with microbial consortia enriched via two steps accumulation method
Karnachuk et al. Bacteria of the sulfur cycle in the sediments of gold mine tailings, Kuznetsk Basin, Russia
Shaaban et al. Removal of heavy metals from aqueous solutions using multi-metals and antibiotics resistant bacterium isolated from the Red Sea, Egypt
Altowayti et al. Significance of bio-treatment by acid washing for enlargement of arsenic desorption in indigenous arsenic-resistant bacteria from gold mine
Jayaraman et al. Metal biosorption by magnetotactic bacteria isolated from fresh water sediments and characterization of extracted magnetosomes
Bhatt et al. Application of genetically modified microorganisms for bioremediation of heavy metals from wastewater
WO2024237867A1 (fr) Utilisation de souches bactériennes résistantes à l'antimoine pour la bioremédiation et procédé de bioremédiation utilisant ces mêmes souches bactériennes
Chen et al. Enhanced sequestration of molybdenum (VI) using composite constructed wetlands and responses of microbial communities

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23728883

Country of ref document: EP

Kind code of ref document: A1