WO2015056185A1 - Complexe microbien utilisable en vue de la séquestration de nitrates et de phosphates à des fins de sauvegarde de l'environnement - Google Patents

Complexe microbien utilisable en vue de la séquestration de nitrates et de phosphates à des fins de sauvegarde de l'environnement Download PDF

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WO2015056185A1
WO2015056185A1 PCT/IB2014/065326 IB2014065326W WO2015056185A1 WO 2015056185 A1 WO2015056185 A1 WO 2015056185A1 IB 2014065326 W IB2014065326 W IB 2014065326W WO 2015056185 A1 WO2015056185 A1 WO 2015056185A1
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nitrate
phosphate
microbial consortium
present
consortium
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PCT/IB2014/065326
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Shaon Ray Chaudhuri
Indranil MUKHERJEE
Ashoke Ranjan THAKUR
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Shaon Ray Chaudhuri
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    • 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
    • C02F3/30—Aerobic and anaerobic processes
    • C02F3/302—Nitrification and denitrification treatment
    • C02F3/305—Nitrification and denitrification treatment characterised by the denitrification
    • 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
    • C02F3/30—Aerobic and anaerobic processes
    • C02F3/308—Biological phosphorus removal
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • C02F3/02—Aerobic processes
    • C02F3/10—Packings; Fillings; Grids
    • C02F3/101—Arranged-type packing, e.g. stacks, arrays
    • 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
    • C02F3/02—Aerobic processes
    • C02F3/10—Packings; Fillings; Grids
    • C02F3/105—Characterized by the chemical composition
    • C02F3/107—Inorganic materials, e.g. sand, silicates
    • 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
    • C02F3/02—Aerobic processes
    • C02F3/10—Packings; Fillings; Grids
    • C02F3/109—Characterized by the shape
    • 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
    • C12R2001/07—Bacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Definitions

  • TITLE MICROBIAL CONSORTIUM FOR NITRATE AND PHOSPHATE SEQUESTRATION FOR ENVIRONMENTAL SUSTENANCE
  • the present invention relates to a microbial consortium comprising a plurality of bacterial strains, capable of sequestering nitrogen and phosphate from biomass, for environmental protection and sustenance.
  • the present invention further provides a method of treatment of wastewater effluent by treating the wastewater effluent with a microbial consortium comprising a plurality of bacterial strains, capable of sequestering nitrogen and phosphate.
  • Wastewater treatment has grown significantly over the past few decades.
  • Environmental protection regulations require treatment of disposable effluent from wastewater prior to its drainage into natural water system.
  • Waste water is contaminated by both organic and inorganic waste compounds.
  • Organic matter is much easier to decompose and remove from the wastewater as compared to the inorganic waste.
  • wastewater contaminated from domestic and industrial sources is chemically treated prior to being discharged into a natural water system.
  • the chemical treatment is much more expensive and further cause greater water quality problems. So to make the water treatment process more economical and safer for drinking, recently the wastewater is treated biologically.
  • Biological treatment means degradation of the impurities with the help of microorganisms/plants/combination of both.
  • Nitrate and phosphate are two major pollutants due to excessive use of fertilizers in agriculture which have emerged as a global problem and its potential threat is marked on the environmental sustenance as well as on the public health. Nitrates are highly water soluble, and so move readily with surface runoff into rivers or with water percolating through the soil profile into the groundwater below. However, phosphate is not water soluble, so it moves only with soil movement, as it adheres to soil particles. [0003] The runoff of nitrate and phosphate into lakes and streams causes accelerated eutrophication. Eutrophication is a natural process that typically occurs as lakes age. However, human-caused, accelerated eutrophication occurs more rapidly, and causes problems in natural water bodies. Eutrophication stimulates the growth of algae causing depletion of oxygen and diminishing the penetration of light which in turn harms the aquatic life in water.
  • Nitrate is also a potential human health threat especially to infants, causing the condition known as methemoglobinemia, also commonly known as "blue baby syndrome".
  • Central Nervous System and Cardiovascular System may also be affected as it can be carcinogenic.
  • Nitrate pollution is also reported from paddy as well as tea cultivation where the nitrate leaching causes acidification of soil and increases intensity of nitrate level in ground water.
  • nitrate could be called as a necessary evil, which is extremely essential for plant growth but when leached into water bodies, causes major disaster for environmental as well as human health.
  • Biological phosphorus removal processes are well known in the art and have been applied to the wastewater for the removal of phosphate.
  • Enhanced biological phosphorus removal (EBPR) is one such process which is a key player in recovering phosphate from waste stream and curbing down the possibilities of eutrophication.
  • EBPR is an essential cost effective step in phosphate removal and recovery compared to the traditional process of phosphate removal by chemical precipitation.
  • the phosphate removing organisms responsible for EBPR have been seen to possess the unique property of polyphosphate accumulation. Previous studies have demonstrated that microorganisms like Aerobacter spp., Pseudomonas spp.
  • Moraxella spp. Escherichia coli, Mycobacterium spp., Beggiatoa spp., Neisseria meningitides, Klebsiella spp., Azotobacter vinelandii, Neurospora crassa are efficient in accumulating and releasing phosphorous. (Tsai et al, 1979; Cramer et al, 1980, Bitton, 1994)
  • Phosphorus removal is effected through its assimilation as microorganisms grow and uptake the inorganic phosphorus in excess which become stored within the cells in the form of polyphosphate granules (volutin).
  • Poly phosphate kinase (PPK) gene, exopolyphosphatase (PPX) gene along with a regulatory site constituting ppk/ppx operon, are primarily responsible for this synthesis of polyphosphate which is a key of phosphate removal and recovery from waste water.
  • Inorganic polyphosphate (polyP) is a rich source of energy. These internal reserves support the prolonged growth and survival of the organism in adversity.
  • PolyP compounds are linear polymers containing tens to hundreds of phosphate residues linked by energy-rich phosphoanhydride bonds. PolyP helps the microorganisms to adapt to extremes of salinity, osmolality, desiccation, UV radiation, pH, and temperature in the environment. In bacteria, polyP levels are controlled by several polyphosphatases as well as by two families of polyP kinases, PPKl and PPK2. The sequences of these enzymes are not related. PPKl synthesizes most of the polyP in the cell, using ATP as a substrate, whereas the only example of PPK2 characterized up to now generates GTP from GDP.
  • the present invention satisfies these needs, as well as others, and efficiently overcomes the deficiencies found in the background art.
  • the present invention relates to a microbial consortium, capable of sequestering phosphate and nitrate from biomass, comprising bacterial strains selected from the group consisting of Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059.
  • the microbial consortium sequesters phosphate and nitrate from wastewater effluent.
  • Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059 are present in equal ratio.
  • microbial consortium of the present invention sequesters 62 to 66% phosphate and sequesters and removes 94.6 to 98.98% nitrate from waste water effluent.
  • the present invention also relates to a method of treatment of wastewater effluent, comprising the steps of :
  • the solid support is a steel mesh.
  • the bioreactor is a packed bed bioreactor.
  • the microbial consortium is capable of preventing eutrophication of water bodies.
  • biomass with sequestered phosphate and nitrate is used as biofertilizer.
  • microbial consortium of the present invention is capable to fasten initiation of flowering in plants.
  • microbial consortium of the present invention is capable to promote plant growth and maintain fertility of soil.
  • Figure 1 illustrates a growth curve for a microbial consortium of the present invention consisting of a combination of MCC0008, MCC2059, and MCC2071, in a 5 litre (L) suspended bed bioreactor.
  • Figure 2a illustrates reduction of concentration of nitrate in media with time.
  • Figure 2b illustrates reduction of concentration of phosphate in media with time.
  • Figure 3a illustrates reduction kinetics of nitrate for a microbial consortium of the present invention consisting of a combination of MCC0008, MCC2059, and MCC2071, in a 5L suspended bed bioreactor.
  • Figure 3b illustrates reduction kinetics of phosphate for a microbial consortium of the present invention consisting of a combination of MCC0008, MCC2059, and MCC2071, in a 5L suspended bed bioreactor.
  • Figure 4a illustrates percentage reduction of nitrate with a reducing concentration of nitrate broth in 9.0 L packed bed reactor
  • Figure 4b illustrates percentage reduction of phosphate with a reducing concentration of nitrate broth in 9.0 L packed bed reactor.
  • Figure 5a illustrates percent reduction in nitrate concentration in 9L bioreactor in batch mode.
  • Figure 5b illustrates percent reduction in phosphate concentration in 9L bioreactor in batch mode.
  • Figure 6a illustrates percent reduction in nitrate concentration in 9L bioreactor in continuous mode.
  • Figure 6b illustrates percent reduction in phosphate concentration in 9L bioreactor in continuous mode.
  • Figure 7a illustrates percent reduction in nitrate concentration in 9L bioreactor in continuous mode with changing flowrate.
  • Figure 7b illustrates percent reduction in phosphate concentration in 9L bioreactor in continuous mode with changing flowrate.
  • Figure 8 illustrates time course of nitrate reduction in a 9L packed bed bioreactor in presence of different concentrations of nitrate in an influent.
  • Figure 9 illustrates TEM images of strains showing vacuoles upon accumulation of nitrate (MCC0008) and phosphate (MCC0008, MCC2071 and MCC2059).
  • Figure 10 illustrates comparison of different parameters reflecting enhanced yield with a microbial consortium of the present invention consisting of a combination of MCC0008, MCC2059, and MCC2071, as compared to Control (no fertilizer) but less than chemical fertilizer application.
  • the present invention relates to a microbial consortium, capable of sequestering phosphate and nitrate from biomass, comprising bacterial strains selected from the group consisting of Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059.
  • the bacterial strains comprising Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059 are deposited at the Microbial Culture Collection, Pune, India.
  • the three bacterial strains are studied at genomic level with draft genome sequence available at GenBank with accession number ANAU00000000, ANFK00000000, ANGA00000000 and are found to be novel.
  • the microbial consortium sequesters phosphate and nitrate from wastewater effluent.
  • the microbial consortium of the present invention can be used to prevent eutrophication of water bodies.
  • the microbial consortium of the present invention can also be used to reduce phosphate and nitrate concentration from agricultural runoff, which promotes plant growth and increase soil fertility.
  • the bacterial strains comprising Bacillus sp MCC0008, Bacillus sp
  • MCC2071 and Bacillus sp MCC2059 are phosphate and nitrate accumulators and reducers. Bacillus sp MCC0008 also has an ability to accumulate nitrate intracellular. The extent of nitrate accumulation by MCC0008 increases with an increase in external concentration of nitrate. The capability of MCC0008 to accumulate nitrate is evident from Example 10 described herein below.
  • the microbial consortium of the present invention facilitates removal of phosphate and nitrate from wastewater effluent, lowering levels to limits which are not harmful to environment.
  • the wastewater effluent treated with the microbial consortium of the present invention comprising of sequestered nitrate and phosphate, can be used as biofertilizer.
  • Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059 are present in equal ratio (1 : 1 : 1).
  • the microbial consortium of the present invention can be grown in nitrate broth under optimum conditions.
  • the microbial consortium can be mixed into wastewater effluent, which further results in decrease in eutrophication phenomenon.
  • a plant grown in a soil mixed with the microbial consortium obtained after treatment of wastewater effluent can result into voluminous yield and growth of the plant with better quality.
  • the microbial consortium prepared in the above defined ratio is used as an inoculum for treating wastewater effluent, specifically for removal of phosphate and nitrate.
  • the present invention also relates to a method of treatment of wastewater effluent, comprising the steps of :
  • the solid support is a steel mesh.
  • the bioreactor is a packed bed bioreactor.
  • the bioreactor of the present invention is adapted to support a biofilm and to enable the biofilm to be brought into contact with the wastewater effluent.
  • the microbial consortium can sequester 62 to 66% phosphate and can remove 94.6 to 98.98% nitrate simultaneously in a biofilm based bioreactor system operating in a continuous mode at 65/75/84 revolutions per minute speed of peristaltic pump within 2 hours.
  • the bacterial strains consisting of MCC0008, MCC2071 and MCC2059 can be mixed together in preferably 1 : 1 : 1 ratio to form the consortium of the present invention.
  • the microbial consortia can be mixed and grown in nitrate broth under optimum condition resulting into phosphate and nitrate sequestering consortium of the present invention.
  • the consortium of the present invention can be mixed into soil resulting into removal of nitrate and phosphate from wastewater, which further results in a decrease in eutrophication phenomenon.
  • a plant grown in soil mixed with the consortium of the present invention can result into voluminous yield and plant growth with better quality.
  • the microbial consortium of the present invention is used for plant growth promoting activity.
  • plant growth-promoting activity encompasses a wide range of improved plant properties, including, for example without limitation, improved nitrogen fixation, improved root development, increased leaf area, increased plant yield, rapid seed germination, increased photosynthesis, or an increased in accumulated biomass of the plant.
  • a microbial consortium of the present invention supply a plant with nutrients and thereby can promote plant growth.
  • biomass comprising sequestered nitrate and phosphate can work as biofertilizer in case of Mung bean (Vigna radiata) resulting in a 10.21% increase in yield per hectare as compared to control set (without fertilizer).
  • the yield in case of chemical fertilizer was 23.02% higher than control.
  • elemental as well as nutritional quality of the seeds was better in case of biofertilizer treated samples as compared to chemical fertilizer treated seed samples.
  • MCC 2071 MCC 2059 and MCC 0008 were mixed in varying ratios as represented in Table 1 and checked for the percentage reduction of nitrate and phosphate in nitrate broth for selection of an optimum combination.
  • the highest percentage reduction of nitrate and phosphate was reported using Combination 7 (MCC 2071 : MCC 2059: MCC 0008 in the ratio of 1 : 1 : 1) as indicated in the Table 2a and Table 2b.
  • the growth of cell, nitrate and phosphate measurements were made using spectrophotometer (Biochrome Libra S70).
  • the packed bed bioreactor system was selected. 1% innoculum was used to allow biofilm development on a steel mesh in a packed bed bioreactor (2.51it and 91it) as per reported protocol.
  • the consortium once developed into a biomass was acclimatized to waste water with a carbon source (0.05% citric acid) selected depending on substrate utilization test performed for the isolates.
  • the column was recharged with fresh medium every 24 hours and the differences in concentration of nitrate and phosphate measured using spectrophotometer (Biochrome Libra S70) between 0 hour and after 24 hours were used to calculate the percentage reduction every 24 hours with or without external aeration.
  • Combination 7 was tested in the packed bed reactor (91it under unadjusted/ anaerobic condition) for reduction of nitrate and phosphate as depicted in Figures 4a, 4b.
  • the nitrate and phosphate reduction in this packed bed reactor turned out to be quite satisfactory.
  • the statistical validation of the data was carried out using t-test as depicted in Table 4a and 4b.
  • Table 4a Table representing statistical validation of the extent of nitrate reduction by the microbial consortium, different within the bioreactor with variable composition of the solution undergoing treatment. The t-test was conducted with following null and alternative hypothesis:
  • Hi Variation is significant 100 34 99% 86.179 2.728 significant
  • Table 4b Table representing statistical validation of the extent of phosphate reduction by the microbial consortium, different within the bioreactor with variable composition of the solution undergoing treatment. The t-test was conducted with following null and alternative hypothesis:
  • null hypothesis Treatment for which t value is significant, null hypothesis is rejected.
  • the 9L bioreactor was operated in continuous mode with canal water containing 0.05% citric acid for 19 days at a flow rate of 40.5 ml/min or 2.43 1/hour (inflow) and an outflow of effluent (treated wastewater with respect to nitrate and phosphate) at the rate of 34 ml/min or 2.041itre/hour with a retention time of 1.93 hours.
  • Table 5 The Flow rate of the influent and effluent as a result of variation in the speed of the peristaltic pump.
  • the consortium of the present invention could be used for both nitrate and phosphate sequestration from waste water, it was tested for PGPB traits and vigour index calculation.
  • the formulation was used for germination trail, pot trial as well as field trial for Vigna radiata cultivation by adding consortium to soil during sowing. Vigna radiata var Samrat (developed by IIPR, Kanpur) was used for the trail from Feb 2013 to May 2013 (Spring Summer Planting).
  • Vigna radiata var Samrat developed by IIPR, Kanpur
  • 14 days experiment was carried out in triplicate. The soaked seeds were sown, followed by formulation application in the soil. The setup was maintained for 14 days while monitoring germination and other growth parameters. The germination index and vigour index were calculated from the data. Pot trial and field trial were carried out.
  • the seeds were lyophilized for 24 hours, manually ground in the mortar and passesl, 0.2gm ground material was pelleted using Pelletizer (Technolab, Kbr Press) at 110 Kg/cm 2 .
  • the mineral content of the pellets were assessed using Energy Dispersive X ray fluorescence (Jordan Valley EX-3600) analysis as per reported protocol.
  • the elemental content has improved post application as compared to control as measured through EDXRF Analysis. This shows that though the yield with chemical fertilizer is higher but the elemental content as compared to control is better in consortium treated seeds while poor following chemical treatment.
  • the microbial consortium of the present invention is by far the fastest nitrate and phosphate sequestering consortium which also has plant growth promoting activity utilizing the sequestered nutrients as fertilizer in case of Mung bean (Vigna radiata).
  • Example 10 Nitrate Accumulation by MCC0008
  • Nitrate accumulation was checked by cell lysis method using sonication. The extent of accumulation per gm of wet weight of pellet was found to be varing between 1278.66 to 1302.12ppm/gm of wet weight (0.021M) of Nitrate using sonication followed by spectrophotometric analysis as per the method of Cataldo as reported in the literature (Cataldo et al. (1975) Rapid colorimetric determination of nitrate in plant tissues by nitration of salicylic acid. Commun. Soil Science and Plant Analysis 6(1) 71-80. DOI: 10.1080/00103627509366547).
  • the strain MCC0008 was subjected to elevated concentrations of nitrate and the corresponding nitrate accumulation was monitored (Table 8). The cell shows higher accumulation when grown in presence of higher concentrations of nitrate.
  • Table 8 Table representing an extent of nitrate accumulation in ppm per gram of wet weight of cells when grown in presence of different concentrations of nitrate in the growth medium
  • the present invention provides a microbial consortium, capable of removing phosphate and nitrate simultaneously by a biomass, comprising of bacterial strains, for environmental protection and agricultural sustenance.
  • the present invention provides a phosphate and nitrate removing microbial consortium wherein the consortium has a capability of plant growth promotion.
  • the present invention provides a phosphate and nitrate removing microbial consortium wherein the consortium fastens initiation of flowering in plants.
  • the present invention provides a phosphate and nitrate removing microbial consortium wherein the consortium prevents eutrophication of water bodies.
  • the present invention provides a phosphate and nitrate removing microbial consortium, wherein the consortium removes as well as sequesters phosphate and nitrate from waste water and biomass and in turn is used as a source of phosphate and nitrate for plant growth promotion.
  • the present invention provides a phosphate and nitrate removing microbial consortium, wherein the biomass comprising sequestered phosphate and nitrate can be used as biofertilizer.
  • the present invention provides a phosphate and nitrate removing microbial consortium wherein the consortium promotes plant growth while constantly maintaining fertility of soil in terms of phosphate and nitrate concentration.
  • the present invention provides a phosphate and nitrate removing microbial consortium wherein the consortium provides better nutritional quality of seeds as compared to chemical fertilizer treated seeds.

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Abstract

La présente invention concerne un complexe microbien, capable de séquestrer phosphates et nitrates, comprenant des souches bactériennes choisies dans le groupe constitué de Bacillus sp. MCC0008, Bacillus sp. MCC2071 et Bacillus sp. MCC2059, et utilisable à des fins de protection et de sauvegarde de l'environnement. La présente invention concerne également un procédé de traitement des eaux usées consistant à traiter les eaux usées au moyen d'un complexe microbien comprenant plusieurs souches bactériennes, capable de séquestrer l'azote et les phosphates. La présente invention concerne en outre l'utilisation, en tant qu'engrais organique, d'une biomasse dans laquelle des phosphates et des nitrates sont séquestrés.
PCT/IB2014/065326 2013-10-16 2014-10-15 Complexe microbien utilisable en vue de la séquestration de nitrates et de phosphates à des fins de sauvegarde de l'environnement WO2015056185A1 (fr)

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