A BACTERIAL CONSORTIUM FOR REDUCING PERCHLORATE AND/OR NITRATE AND THE PROCESS THEREOF

Abstract

The present invention discloses a novel microbial process for decontaminating (per)chlorate and/or nitrate containing matrices. A heterotrophic mixed microbial culture expressing the functional genes responsible for (per) chlorate and nitrate reduction is the major component of the process. The present process can be a better substitute for existing processes for decontaminating perchlorate contaminated propellant wastewater, ion exchange resin/regenerate solutions, etc. The consortium consists or comprises of Halomonas sp. NIIST-PRB-02 (MTCC No. 5911), Bacillus sp. NIIST-PRB-03 (MTCC No. 5912) and Serratia marcescens strain NIIST5 (MTCC No. 5821).

Claims

1. A bacterial consortium useful for detoxifying (per)chlorate and/or nitrate essentially consisting of Halomonas sp. NIIST-PRB-02 (MTCC No. 5911), Bacillus sp. NIIST-PRB-03 (MTCC No. 5912) and Serratia marcescens strain NIIST5 (MTCC No. 5821).

2. A bioprocess for detoxifying (per)chlorate and/or nitrate containing matrices using a bacterial consortium, said process comprising the steps of: a. maintaining a bacterial consortium and providing substrate/nutrients and conditions for sustaining the microbial activity; b. contacting (per)chlorate and/or nitrate contaminated matrix with the bacterial consortium at redox potential below −200 mV, neutral pH and 30±5° C. for a period ranging between 3 to 36 h to obtain detoxified matrices.

3. The bioprocess as claimed in claim 2, wherein the bacterial consortium is facultative anaerobic and essentially consisting of bacteria belonging to genii Serratia, Halomonas and Bacillus isolated from salt adapted sludge from a bioreactor having Accession No. MTCC No. 5911, MTCC No. 5912, and MTCC No. 5821.

4. The bioprocess as claimed in claim 2, wherein the bacteria are maintained in suspension or attached to a suitable inert surface like glass, plastic or ceramic for sustained activity.

5. The bioprocess as claimed in claim 2, wherein (per)chlorate and/or nitrate containing matrices in propellant wastewater containing perchlorate, ammonia, high salt and acidic or alkaline pH or solid matrices having salinity up to 15% and pH in the range 4 to 10 and perchlorate up to 5 g/L can be decontaminated.

6. The bioprocess as claimed in claim 2, wherein (per)chlorate contaminated soil leachate can be treated with this consortium.

7. The bioprocess as claimed in claim 2, wherein the nutrient in step (a) is mineral salt medium containing (g/L water) K.sub.2HPO.sub.4-0.22, KH.sub.2PO.sub.4-0.22, (NH.sub.4).sub.2SO.sub.4-0.22, MgSO.sub.4.7H.sub.2O-0.05, CaCO.sub.3-0.005, FeSO.sub.4.7H.sub.2O-0.005, KClO.sub.4-1.5 g/L and trace metal solution-1.0 ml.

8. The bioprocess as claimed in claim 2, wherein the consortium can reduce perchlorate in the range of ≥5 g/L and nitrate in the range of ≥5 g/L.

9. The bioprocess as claimed in claim 8, wherein reduction of nitrate and perchlorate using mixed bacterial culture was carried out optionally in the presence of ammonium.

10. The bioprocess as claimed in claim 1, wherein the bacterial consortium was contacted with (per) chlorate and/or nitrate at a concentration of ≥5 g/L.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1. Agarose gel image showing A) 100 bp ladder (lane 1), amplified per A (lane 2); B) 100 bp ladder (lane 1), amplified Cld gene (lane 2); C) 100 bp ladder (lane 1), amplified narG gene (lane 2).

[0038] FIG. 2. Perchlorate reduction profile of diluted industrial effluent containing different initial ClO.sub.4.sup.− concentration at 5% NaCl by the present consortium.

[0039] FIG. 3. Schematic representation of the process for decontaminating (per)chlorate and/or nitrate contaminated water (A) bioreactor with suspended biomass; (B) fixed film type bioreactor.

[0040] FIG. 4. Reduction of perchlorate alone and perchlorate-nitrate mixture by the present consortium in batch culture.

[0041] FIG. 5. Reduction of perchlorate in presence of ammonium at different initial concentration in batch culture.

[0042] FIG. 6. Reduction profile of perchlorate in a mini-fermenter using the consortium developed under aerobic conditions.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention relates to a novel bioprocess for detoxifying (per)chlorate and/or nitrate containing matrices using a mixed bacterial culture dominated by genii belongs to—Halomonas sp. NIIST-PRB-02 (MTCC 5911), Bacillus sp. NIIST-PRB-03 (MTCC 5912) and Serratia marcescens strain NIIST5 (MTCC 5821) under specified conditions.

[0044] Through this process, liquids or solid matrices containing (per) chlorate and/or nitrate at wide concentration ranges and under high salt and wide pH levels can be decontaminated.

[0045] This process can find application for treating perchlorate and nitrate contaminated water, propellant wastewater, ion exchange resin regenerate solution, etc.

[0046] The present invention discloses a novel bioprocess for detoxifying (per)chlorate and/or nitrate containing solutions or solid matrices using a mixed bacterial culture (consortium) dominated by bacteria essentially consisting of genii Halomonas, Bacillus and Serratia. This invention comprises mainly two components, (1) the specific bacterial consortium and (2) the process for decontaminating (per) chlorate and/or nitrate contaminated matrices. The following description addresses both.

(1) Bacterial Consortium Reducing (Per) Chlorate and/or Nitrate:

[0047] The present consortium consists mainly of three bacteria belongs to genii Halomonas, Bacillus and Serratia. The cultures of these three strains were deposited at MTCC with the following details—Halomonas sp. NIIST-PRB-02 (MTCC 5911), Bacillus sp. NIIST-PRB-03 (MTCC 5912) and Serratia marcescens strain NIIST5 (MTCC 5821). All three bacteria in the consortium can be cultured and maintained together or individually. All the bacteria expresses the genes specific for the reduction of (per) chlorate and/or nitrate, so that this consortium can be effectively used for treating liquid/solid matrices contaminated with (per)chlorate and/or nitrate. This consortium is facultative anaerobic so that it can work under both aerobic and anoxic condition.

[0048] The consortium can be applied for (per)chlorate and nitrate reduction in batch cultures or in continuously fed bioreactors. Moreover, the consortium can be maintained in an engineered biological system (bioreactor) either as suspended biomass or attached to inert substrates to form a fixed film. The consortium can tolerate high concentration of perchlorate (up to 5.0 g/L), high salt (>15%), high ammonium (0.5 g/L) and extreme pH (4-9) which makes it ideal for decontaminating ion exchange regenerate solution.

[0049] The consortium utilizes organic substrates for growth including sugars, organic acids, alcohols etc. However, it prefers acetate as the electron donor for reducing perchlorate into chloride and oxygen.

(2) Process for Reducing (Per)Chlorate and/or Nitrate:

[0050] In the present process, (per)chlorate and/or nitrate contaminated matrix (liquid or solid) will be exposed to the above described bacterial consortium for a specific period of time under anoxic environment in a suitable bioreactor system.

[0051] To support the bacterial activity an organic substrate and nutrients will be provided to the reactor. Under such condition the consortium will use organic substrate as electron donor and (per)chlorate and/or nitrate as electron acceptor and reduces the latter into non-toxic chloride, oxygen or nitrogen.

[0052] In this process the bacterial system can be maintained either as suspended (fluidized) biomass or fixed film on inert substrates like plastic, clay, glass etc.

The process can be used for treating wastewater or solutions or solid matrix contaminated with (per) chlorate and/or nitrate.

[0053] Thus the Present invention provides bacterial consortium useful for detoxifying (per) chlorate and/or nitrate essentially consist of Halomonas sp. NIIST-PRB-02 (MTCC 5911), Bacillus sp. NIIST-PRB-03 (MTCC 5912) and Serratia marcescens strain NIIST5 (MTCC 5821).

[0054] In an embodiment, a bioprocess for detoxifying (per)chlorate and/or nitrate containing matrices using a bacterial consortium wherein the said process comprising the steps of: [0055] a. maintaining the bacterial consortium and providing substrate/nutrients for sustaining the microbial activity in a bioreactor; [0056] b. contacting (per)chlorate and/or nitrate contaminated matrix with the bacterial consortium in the bioreactor of step (a) at redox potential below −200 mV, neutral pH and 30±5° C. for a period ranging between 3 to 12 h to obtain detoxified matrices.

[0057] In yet another embodiment, in a process, bacterial consortium of bacteria belongs to genii Serratia, Halomonas and Bacillus isolated from salt adapted sludge from a bioreactor and has been deposited with MTCC having Halomonas sp. NIIST-PRB-02 (MTCC 5911), Bacillus sp. NIIST-PRB-03 (MTCC 5912) and Serratia marcescens strain NIIST5 (MTCC 5821).

[0058] In yet another embodiment, bacteria are maintained in suspension or attached to a suitable inert surface like glass, plastic or ceramic for sustained activity.

[0059] In yet another embodiment, wherein (per)chlorate and/or nitrate containing matrices in propellant wastewater containing perchlorate, ammonia, high salt and acidic or alkaline pH or solid matrices having salinity up to 15% and pH in the range 4 to 10 and perchlorate up to 5 g/L can be decontaminated.

[0060] In yet another embodiment, nutrients in step (a) is mineral salt medium containing (g/L water) K.sub.2HPO.sub.4-0.22, KH.sub.2PO.sub.4-0.22, (NH.sub.4).sub.2SO.sub.4-0.22, MgSO.sub.4.7H.sub.2O-0.05, CaCO.sub.3-0.005, FeSO.sub.4.7H.sub.2O-0.005, KClO.sub.4-1.5 g/L and trace metal solution-1.0 ml.

[0061] In yet another embodiment, reduction of perchlorate is in the range of ≥5 g/L.

[0062] In yet another embodiment, reduction of nitrate is in the range of ≥5 g/L.

[0063] In yet another embodiment, reduction of nitrate and perchlorate using mixed bacterial culture was carried out optionally in the presence of ammonium.

[0064] In yet another embodiment, reaction using bacterial consortium was carried out in the presence of (per)chlorate and/or nitrate ≥5 g/L concentration.

The following examples serve to provide the best modes of practice for the present invention, and should not be constructed as limiting the scope of the invention:

Example 1: The Bacterial Consortium; its Characteristics, Preparation and Maintenance

[0065] The bacterial consortium (mixed culture) used in the present process was dominated by bacteria belongs to Halomonas sp. NIIST-PRB-02 (MTCC 5911), Bacillus sp. NIIST-PRB-03 (MTCC 5912) and Serratia marcescens strain NIIST5 (MTCC 5821). All three bacteria were isolated from a laboratory scale bioreactor for reducing (per)chlorate and/or nitrate.

[0066] The consortium can be grown and maintained in mineral salt medium containing (g/L water) K.sub.2HPO.sub.4-0.22, KH.sub.2PO.sub.4-0.22, (NH.sub.4).sub.2SO.sub.4-0.22, MgSO.sub.4.7H.sub.2O-0.05, CaCO.sub.3-0.005, FeSO.sub.4.7H.sub.2O-0.005, KClO.sub.4-1.5 g/L and trace metal solution-1.0 ml. A higher perchlorate to acetate ratio like 1:2 was required for complete reduction of (per)chlorate and/or nitrate. The consortium can be maintained under both aerobic and anoxic conditions.

Under optimum conditions of pH (.sup.˜7), temperature (30±3° C.) and anoxic condition, the specific growth rate (μ) of the consortium was 0.025/h in the above media. Alternatively, the consortium can be developed and maintained in nutrient broth or Luria Bertani media.

[0067] The consortium was screened for its potential to reduce perchlorate and/or nitrate. This was done by analyzing the presence of metabolic genes responsible for perchlorate and/or nitrate. Polymerase chain reaction (PCR) with specific primers for perchlorate reductase (perA gene) and chlorite dismutase (cld gene) involved in the complete degradation of perchlorate was done. The gene product obtained was sequenced and it was confirmed through similarity check using BLAST (NCBI) algorithm. Similarly the presence of nitrate reductase (narG) gene was also confirmed. The PCR images were presented in FIG. 1.

[0068] The present consortium was able to reduce both perchlorate and nitrate simultaneously under anoxic condition using acetate as substrate (electron donor). It was able to tolerate and reduce perchlorate at ppb (μg/L level) to g/L level.

Example 2: Reduction of Perchlorate in Discharge Brine Using the Consortium in Batch Culture

[0069] The present consortium was tested for reducing perchlorate present in an industrial discharge brine (effluent). Effluent collected from an ammonium perchlorate (rocket fuel) manufacturing unit was used for the treatment study. The effluent had perchlorate-70 g/L, ammonium-17.9 g/L, salinity 5%, pH-6.0 and TOC-9.46 mg/L. This effluent was diluted with water to bring down the perchlorate and ammonium concentration as well as salt level.

[0070] Batch experiment was conducted with diluted industrial brine (ClO.sub.4.sup.− at 100-500 mg/L level). The consortium acclimatized to 5% NaCl was used for the treatment study. This was done by slow adaptation of the consortium to high salt conditions. The batch experiment was conducted in 25 ml capacity crimp cap vials. The mineral medium as described in example 1 (except the level of KClO.sub.4 and CH.sub.3COONa), was used in the experiment. Dissolved oxygen in the medium was expelled by sparging air free nitrogen from a cylinder filtered through 0.22 μm filters (Millipore) for 10 min. The vials were incubated under at 30±3° C. for a period of 72 h

[0071] The consortium from an anoxically grown culture at late log phase was pelletized (8000×g), washed and resuspended in deaerated mineral medium in the vial (initial OD.sub.600 0.03-0.05). The vials were incubated under at 30±3° C. and samples were withdrawn using a syringe every 12 h for estimating ClO.sub.4.sup.−, Cl.sup.− and OD.sub.600.

[0072] Perchlorate and chloride were estimated through IC (Dionex-1100) with AS 11 column and anion self-regenerating suppressor (ASRS 300, 4 mm) with NaCO.sub.3:NaHCO.sub.3 (1:4.8 mM) buffer as mobile phase at flow rate of 1.5 mL/min.

[0073] Reduction of perchlorate present in an industrial discharge containing high salt and ammonia is presented in FIG. 2. It is demonstrated that the present consortium can be used for treating perchlorate contaminated effluents under high salt and in presence of ammonium.

Example 3: Decontamination of Perchlorate in Water Using the Consortium in Continuous Bioreactors

[0074] A process flow chart for decontaminating perchlorate in a contaminated aqueous system using the present bacterial consortium is presented in FIG. 3. The bacterial consortium was maintained as a suspended biomass in a 3 Lit capacity glass tank (working volume 2.5 lit). Totally anoxic atmosphere inside the reactor was created initially by sparging with air free nitrogen for 10 min. The biomass load level in the reactor was equivalent to around 10 g dry weight. Slow mixing of the biomass was done using an impeller. The temperature in the reactor was at 30±3° C. The bacterial growth inside the reactor was supported through providing a nutrient solution as specified in example 1.

[0075] Water containing perchlorate at 10 mg/L was stored in a plastic reservoir. It was introduced into the bioreactor using a peristaltic pump (Watson-Marlow). The nutrient solution was mixed with the influent before introducing into the reactor. The perchlorate contaminated water during its retention inside the reactor will be used by the consortium and reduces it into non-toxic chloride and oxygen. The treated effluent will be collected and it was analyzed for residual perchlorate and chloride build up. Perchlorate and chloride were analyzed using a DIONEX ion chromatographic system using DIONEX AS16 column, anion suppressor and 50 mM NaOH mobile at 1 ml/min. During this experiment, complete reduction of perchlorate was observed.

[0076] Alternatively, a fixed film type bioreactor was also used for treating perchlorate contaminated water. An acrylic column of 4.5 cm internal diameter and 60 cm height was used for making the bioreactor. Up to 40 cm height, inert media like plastic rings or glass beads was used as surface for the biofilm growth. The reduction of perchlorate was similar to the suspended growth system.

Example 4: Simultaneous Reduction of Perchlorate and Nitrate by the Consortium

[0077] The simultaneous reduction of nitrate and perchlorate by the present consortium was demonstrated in batch cultures. The experiment was conducted in 25 ml capacity crimp cap vials. The mineral medium as described in example 1 (except the level of perchlorate and acetate), was used in the experiment. Dissolved oxygen in the medium was expelled by sparging air free nitrogen from a cylinder filtered through 0.22 μm filters (Millipore) for 10 mins. The consortium from an anoxically grown culture at late log phase was pelletized (8000×g), washed and resuspended in deaerated mineral medium in the vial (initial OD.sub.600 0.03-0.05). The vials were incubated under at 30±3° C. and samples were withdrawn using a syringe every 12 h for a period of 72 h for estimating ClO.sub.4.sup.−, Cl.sup.− and OD.sub.600. Perchlorate and chloride were estimated through IC (Dionex-1100) with AS 11 column and anion self-regenerating suppressor (ASRS 300, 4 mm) with NaCO.sub.3:NaHCO.sub.3 (1:4.8 mM) buffer as mobile phase at flow rate of 1.5 mL/min.

[0078] Nitrate (as KNO.sub.3; 10 mg/L NO.sub.3.sup.−) was added to the inorganic mineral medium containing perchlorate and acetate (10 mg/L and 20 mg/L respectively). Nitrate free positive control and heat killed (non biological) controls were run parallel. It was found that both nitrate and perchlorate were reduced simultaneously. However, nitrate was reduced more rapidly compared with perchlorate. The result of the present study is given in the FIG. 4.

Example 5: Perchlorate Reduction in Presence of Ammonium

[0079] The present consortium was tested for perchlorate reduction activity in presence of ammonium at various initial concentrations. The experiment was conducted in 25 ml capacity crimp cap vials using the mineral salt medium as described in example 1. Ammonium was added to the medium as (NH.sub.4OH) at different levels to bring the final ammonium concentration in the range 100-800 mg/L. pH of the medium was subsequently adjusted to neutral range by adding 0.1M HCl. The vials were incubated under at 30±3° C. for a period of 72 h and samples were withdrawn using a syringe every 12 h for estimating ClO.sub.4.sup.−. Perchlorate was estimated through IC (Dionex-1100) with AS 11 column and anion self-regenerating suppressor (ASRS 300, 4 mm) with NaCO.sub.3:NaHCO.sub.3 (1:4.8 mM) buffer as mobile phase at flow rate of 1.5 mL/min.

[0080] A significant ClO.sub.4.sup.− reduction (above 85%) was observed at NH.sub.4.sup.+ concentrations up to 500 mg/L. However further increasing ammonium to 800 mg/L resulted in decline in reduction to 41%. The result of the study is presented in FIG. 5.

Example 6: Facultative Anaerobic Consortium Capable of Reducing Perchlorate

[0081] The facultative anaerobic nature of the present consortium was tested in a 500 ml capacity mini-fermenter (Applikon). The consortium from a stock was inoculated in Nutrient broth medium (100 ml) in a 500 ml conical flask and incubated under aerobic conditions in a rotary shaker (150 rpm) for 48 h under ambient temperature. After 48 h the pellet was separated by centrifugation, washed with sterile mineral salt medium and re-suspended in the mineral salt medium which is used as the inoculum (50 ml). Around 400 ml of mineral salt medium added with perchlorate at 10 mg/L level and acetate at 30 mg/L was taken in the fermenter vessel and incubated under anoxic condition with continuous mixing (100 rpm) and at 30° C. Dissolved oxygen and pH are continuously monitored. Samples were withdrawn very 12 h to estimate residual perchlorate in the medium. It was observed the dissolved oxygen in the medium dropped to zero in <12 h and subsequently perchlorate reduction initiated slowly, and perchlorate level came to undetectable level after 72 h. The initial lag in reduction of perchlorate was due to the presence of residual oxygen in the medium. The bacterial cells start consuming perchlorate when dissolved oxygen is completely exhausted. In the present experiment the consortium was developed under aerobic condition, its perchlorate reduction property was not found affected when it was subsequently incubated under anoxic condition (facultative anaerobic). The data of the experiment is presented in FIG. 6.

Major Advantages of the Invention:

[0082] 1. The present process using the mixed microbial system can be used for decontaminating perchlorate in wide concentration range from ppb (microgram/L) level to g/L level. [0083] 2. The mixed microbial system in the present process for decontaminating perchlorate can tolerate ammonia, so that it can be used for treating propellant wash water containing perchlorate and ammonia. [0084] 3. The present process using the mixed microbial system can be used for decontaminating wastes containing simultaneously (per)chlorate and/or nitrate. [0085] 4. The present process can be used for treating (per)chlorate and/or nitrate containing solutions at wide range of pH (acidic to alkaline) and high salt conditions. [0086] 5. The mixed microbial culture in the present process is facultative anaerobic so that it can be maintained under aerobic and anoxic conditions.