Specification
TECHNICAL FIELD OF THE INVENTION
The present invention relates to myco-capsules for bioremediation of waste water. Particularly, the invention relates to a method of preparation of microbial culture and its encapsulation along with the nutrient requirement inside the capsule called myco-capsule. Such myco-capsules eliminate the need for the additional nutritional requirement for the growth of the microbes during bioremediation of the waste water contaminated with dyes and/or metals. The myco-capsules of the present invention provide enhanced shelf-life, easy storage and transportation of the formulations of fungal strains at ambient conditions without exhibiting any substantial loss in viability and pollutant removing ability.
BACKGROUND OF THE INVENTION
Waste water stream from various industries like dying, textile, paint and electroplating are contaminated with the mixture of heavy metals, dyes and other organic/inorganic compounds. Synthetic dyes present in the industrial effluents are recalcitrant organic compounds. On the other hand, heavy metals are non-degradative in nature and produce various health hazards. Specialized microbial cultures possess the ability for bioremediation of such dyes, heavy metals and/or both. However, a suitable storage and delivery system for bioaugmentation of waste water streams with selected cultures is required.
Various treatment methods have been adopted for the removal of contaminants from the wastewater using microbes. US5476788 A8 (Lamar et al.) and US4891320 A (Aust et al) deal with the lignin degrading fungi, which can degrade the halogenated hydrocarbons present as contaminants. However the fungi were not encapsulated and require nitrogen limited conditions for the degradation process.
WO 2014057443 A2 describes the encapsulation of the viable micro-organism which can leach the heavy metal from the waste water. But, here encapsulated formulation requires acidic pH ranging from 1-2 for activity.
US5084389 A describes encapsulated bio-sorption composition as bio-sol for heavy metal removal from the water. But the process for encapsulation requires micro-organism, matrix
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compound and a metal compound. Similarly, US8367109 B2 describes the encapsulation of the micro-organism within polymer fiber for heavy metal remediation. However the encapsulated micro-organism has shelf life of only 2 months at -70ºC.
Patent US20140342437 A1 describes the encapsulation of the bacillus sp. for the removal of organic contaminants, TSS, oil, Fats etc. from the waste water. But process of formulation requires fermentation, drying and grounding to the fine powder with dextrose monohydrate as the carrier.
Some researchers have been producing myco-granules and invert emulsion having bio-pesticidal activities for field applications (Pereira et al. 1990, Batta et al 2003 and Connick et al 1991). Myco-granules can greatly ease the handling and dosing problems. But storage of myco-granules is a big problem as it requires 4 ºC temperatures in many cases to remain active for the particular application. Moreover, it is susceptible to cross contamination with undesirable microbes.
The suitable commercial formulation should be cheap, quick and easy to produce. It should provide adequate protection against cross contamination from other microbes. Developing an economical method for stable microbial propagules in mass is critical step in commercialization of the product (Bower, 1982). In this connection, several low cost substrates have been attempted. Booth et al. (1988) reported the formulation of three entomopathogenic fungi (Metarhizium anisopliae, Beauveria bassiana and Paecilomyces farinosus) that was prepared using long grain cooked rice against certain pests. The number of conidia sporulated from the formulation did not decline between 4 and 20 days at 4ºC and 20ºC, respectively. The extent of sporulation by a fungus is affected by the media used and its C/N ratio (Gao et al., 2007). Moreover, the extent of sporulation is strain dependent also, for example, Metarhizium sp. produces maximum sporulation at 160:1 C/N ratio whereas Paecilomyces sp. produces maximum sporulation at 10:1 C/N ratio.
Numerous solid matrixes with several combinations such as wheat bran–sand mixture, sawdust–sand–molasses mixture, corn cob–sand–molasses mixture, bagasse–sand–molasses mixture, organic cakes, cowdung–sand mixture, compost/farm manure, have been tested to prepare
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granular/powder formulations of bio-control agents. Khan et al.(2011) prepared the formulation of fungi (T. harzianum and P. chlamydosporia) and bacteria (B. subtilis and P. fluorescens) as biocontrol agent having shelf life of 32 weeks. Abraham et al. (2014) used saw dust, soil and 5% molasses (15:5:1) for mass production of bacterial and fungal consortium. This powdered consortium was able to degrade endosulfan. The shelf life of the consortium was checked upto 12 weeks only.
Stirling et al.(1998) prepared granule formulation of fungi Verticillium chlamydosporium using carrier (Kaolin) and Binder (gum arabic) against a pest, Meloidogyne javanica . The fungus grew vigorously from these granules when they were placed on agar and retained its viability when granules were stored in vacuum-sealed bags at 25°C for 12 months. Connick et al. (1991) produced fungal propagules encapsulated in wheat gluten matrix as myco-herbicide. This myco-herbicide is able to show viability even after 18 months of storage at 4ºC.
Although enormous research has been carried out, no formulation has been reported for the dual purpose of dye and/or metal removal that can be stored and transported at ambient conditions without the loss of viability and pollutant removing ability. Particularly, there have been no reports for the formulation of fungal strains having a good shelf life at ambient conditions for removal of dyes and/or metals from the waste water.
OBJECTIVES OF THE INVENTION
The main objective of the present invention is to provide myco-capsules for bioremediation of waste water.
Another objective of the present invention is to provide myco-capsules comprising fungal strains and nutrient media specific for the fungal strain inside the myco-capsule only.
Another objective of the invention is to produce color coded myco-capsules to target specific application i.e. metal removal, dye removal or both metal-dye removal in the waste water.
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Another objective of the invention is to provide a formulation of fungal strains having enhanced stability at ambient conditions without loss in viability and pollutant removal efficiency of the fungal strain.
Another objective of the invention is to provide a stable formulation of fungal strains for bioremediation of waste water for metal and/or dye removal.
Still another objective is to provide a method of preparation of myco-capsules for bio remediation of waste water comprising metal and/or dye.
Still another objective is to provide a method for bioremediation of waste water using myco-capsules.
The foregoing has outlined some of the pertinent objectives of the invention. These objectives should not be construed to be merely illustrative of some of the more prominent features and applications of the intended invention. Many other beneficial results can be obtained by applying the disclosed invention in a different manner or modifying the invention within the scope of disclosure.
SUMMARY OF THE INVENTION
The present invention relates to myco-capsules for bioremediation of waste water containing heavy metals and dyes. The treatment of mixed waste water is brought about by myco-capsules which are carrier based encapsulated formulations of fungal strains. The present application also discloses method of preparation of myco-capsules whereby the media specific for the encapsulated fungal strain is also packed inside the capsule and a barrier is used to separate the media and the fungal strain inside the myco-capsule. The presence of inherent media inside the myco-capsules eliminates the need for providing nutritional requirements during the treatment of waste water. In addition, multiple strains in one capsule can be incorporated for better efficacy during the bioremediation process and for simultaneous removal of dye and metal.
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Furthermore, to make dye and metal removal process easy, color coding of the myco-capsule has been disclosed in the present invention. The particular color coded capsule contains the specific strain as well as optimized media having water soluble barrier separating the both. Hence, these color coded myco-capsules can be used for particular application according to the need with ease. Nevertheless, no microbial formulation in the form of capsule has been reported for dual purpose of dye and metal removal or both simultaneously. Moreover, myco-capsules of the present invention provide protection against cross contamination from other microbes and the formulation developed in the form of capsule is useful for easy storage and transportation and is a user friendly formulation.
According to one aspect, the present invention provides a myco-capsule for bioremediation of waste water comprising:
a first part comprising a carrier based formulation of a fungal strain;
a second part comprising media specific for the fungal strain; and
a barrier for separating the first part and the second part,
wherein said first part is fitted into said second part and the barrier is inserted between the first part and the second part to separate the fungal strain and the media in the myco-capsule.
In another aspect, the present invention provides a method of preparation of myco-capsule comprising:
a. inoculating and incubating a fungal strain on a carrier to obtain a powdered formulation of the fungal strain;
b. filling the powdered formulation of the fungal strain in a first part of a pre-fabricated capsule;
c. filing a media specific for the fungal strain in a second part of the capsule; and
d. joining the first part with the second part and inserting a barrier to separate the first part and the second part to obtain the myco-capsule.
In still another aspect, the present invention provides a method for bioremediation of waste water comprising adding myco-capsules into waste water; said myco-capsule comprising a fungal stain
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specific for a specific contaminant in the waste water, a media specific for the fungal strain, and a barrier separating the media and the fungal strain in the myco-capsule; and said waste water comprising heavy metals or dyes or a combination thereof; wherein dissolution of the capsule and the barrier takes place after addition in waste water thereby releasing the fungal strain and the media from the myco-capsules into the waste water; said media providing the required nutrition for the fungal strain to carry out remediation of the waste water.
These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description, accompanying drawings and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
The above and other features, aspects, and advantages of the subject matter will be better understood with regard to the following description, appended claims, and accompanying drawings where:
Figure 1 shows biomass production and dye removal by myco-capsule for (a) Acid Blue 161 and (b) Pigment Orange 34 (concentration: 100 mg L-1; Bar shows biomass and line shows dye removal).
Figure 2 shows biomass production and metal removal efficiency by myco-capsule (a) Cu (II) concentration 100mgL-1 (b) Cr (VI) concentration 100mgL-1. (Bar shows biomass and line shows metal removal).
Figure 3 shows removal kinetics of Cr (VI) by myco-capsule of single strain versus multiple myco-capsules after one year storage at 30 °C (initial Cr (VI) concentration: 100 mg L-1; pH: 6.5±0.2; 30 °C; 150 rpm).
Figure 4 illustrates metal removal by (A) Composite media (CM1) added externally (B) Composite media (CM2) incorporated in the capsule with myco-granules separated by a barrier, in synthetic waste water [100mg L-1 initial Cu (II) concentration; incubated at 30ºC with 150 rpm].
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Figure 5 shows Pollutant removal by various color coded A. lentulus myco-capsules targeted for different pollutants i.e. dye, metal or metal-dye combinations (at 30 ºC with 150 rpm).
Figure 6 shows Viability of different formulations in term of biomass production by (a) A.lentulus (b) A. terreus (Cr(VI) concentration: 100mg L-1 ; at 30 ºC; 150 rpm).
Figure 7 shows comparison of Efficiency of myco-tablets and myco-capsules of A. lentulus in term of (a) Acid Blue 161 dye removal (b) Cr (VI) removal (initial concentration: 100mg L-1 ; at 30ºC; 150 rpm).
DETAILED DESCRIPTION OF INVENTION
The present invention discloses development of myco-capsules having carrier based encapsulated formulations of fungal strains for the treatment of mixed waste water comprising heavy metals and dyes. The treatment of mixed waste water is brought about by myco-capsules which are carrier based encapsulated formulations of fungal strains selected from commonly occurring Aspergillus sp., Rhizopus sp., and Beauveria sp.
In the present disclosure, Aspergillus lentulus, Aspergillus terreus, and Rhizopus oryzae were used as exemplary fungal strains to produce myco-capsules. Aspergillus lentulus was obtained from a textile industry at Badri (Himachal Pradesh). Further, Aspergillus terreus and Rhizopus oryzae were obtained from a waste dumping site in IIT Delhi, New Delhi. The method comprises producing myco-granules of the three fungal strains of Aspergillus lentulus (accession no: FJI72995), Aspergillus terreus (accession no: KC354516), and Rhizopus oryzae (accession no: KC354517) using a previously disclosed methodology (Indian Patent Application no. 2590/DEL/2012). Briefly, each of the fungal strain was inoculated and incubated on a carrier. The carrier is an organic base substrate selected from the group consisting of Maize flour (MF), Rice flour (RF), Wheat flour (WF), or any other Organic bases having C/N ratio ranging from 23-32. The fungal strain was propagated on the carrier to obtain a powdered formulation of fungal strains called myco-granules. Such myco-granules of fungal strains were used to develop an encapsulated formulation called myco-capsules.
Myco-capsules were produced by packing (filling) myco-granules inside pre-fabricated capsules under sterile conditions (0.1 -0.7 g per capsule when maximum capacity is 0.7 g per capsule).
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The myco-capsules comprise spores of a fungal strain in the range of 6.8 x 109 to 4.76 x 1010 CFU. The pre-fabricated capsules used are gelatin capsules (Gelatin ~ 80% to 85%, Moisture ~ 15, Other excipients ~ 0% to 5%; Size of capsule was “00”) having a first part and a second part; wherein first part is smaller than the second part and fits into the second part. The powdered formulation (myco-granules) of the fungal strain is filled in first part of a pre-fabricated capsule and the media (in powdered form) specific for the fungal strain in a second part of the capsule. The ratio of the mycogranules in the first part and the media in the second part is in the range of 0.1-0.3:0.6-0.4 on dry weight basis. A barrier is also used in the myco-capsule for separating the first part and the second part. The barrier is made up of Gelatin (Gelatin ~ 80% to 85%, Moisture ~ 15%, other excipients ~ 0% to 5%) with a thickness of 100μm and dissolution time of 1.5-2.0 min. The myco-capsule is formed by joining the packed first part with the packed second part and inserting the barrier to separate the first part and the second part. There is no loss of viability during myco-capsule production as the process of production does not involve application of pressure, heat or addition of other ingredients and can be produced under sterile conditions.
Dispersion test was conducted on myco-capsules and it was found that complete dissolution of capsule wall and release of granulated formulation into the water takes place within 1.5 to 2 minutes. Further, the myco-capsules were evaluated in terms of their shelf-life (biomass production) and pollutant removal efficiency (removal of heavy metals e.g. Cu(II), Cr(VI), Ni(II), Cd(II), Pb(II), Zn(II), etc.; and dyes such as textile dyes namely Acid Navy Blue, Acid Blue161 and Pigment Orange 34, Acid Red 88, Basic Blue 9, Basic Violet 10, etc.) over a period of one year. It was observed that the metal and dye removal performance of myco-capsules for all the three fungal isolates was stable for the initial six months of storage beyond which it started declining. Further, it was also observed that the combined use of all the three myco-capsules improved the pollutant removal rates. This was demonstrated by employing three myco-capsules simultaneously (in the form of consortia) for Cr (VI) removal. Cr (VI) removal has been taken as an exemplary heavy metal as it is one of the highly toxic metal posing threat to health and the environment. The results showed that consortium accounted for complete removal of Cr(VI) in 72 h, while individual myco-capsules, were unable to remove Cr(VI) even after 120 h, except A.terreus. Further, the Aspergillus sp. based myco-capsules had better performance for removal of metals and metal containing dye while Rhizopus based myco-capsules were better for
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non-metallic dyes. Hence, the combination and dosage of myco-capsules can be varied and optimized as per the concentration of different pollutants in the waste water and myco-capsule specializing in the removal of a particular heavy metal and textile dye may be developed for the required removal of contaminants. Myco-capsules are easy to store and transport formulations of fungal strains and do not require refrigeration. It was observed that myco-capsules can be stored up to one year without exhibiting any substantial loss in viability and pollutant removing ability. Therefore, the formulation of fungal strains in the form of myco-capsules provides advantages over the known methods of bioremediation since storage stability is challenging for microbial formulations.
Earlier, the inventors of the present invention have developed myco-tablets for dye removal (Indian Patent Application no. 2590/DEL/2012), which showed removal of variety of dye solutions containing individual dyes. In the present invention, the inventors developed myco-capsule to target multiple pollutants like metal and/or dyes. It was observed that viability loss during production of myco-tablets (due to pressure during tableting) could be minimized by formulating the myco-granules of fungal strains in the form of myco-capsules. Further, myco-capsules can be color coded very easily for the particular pollutant and the provision of water soluble barrier that allows packaging of both bioactive mycograules as well as the specific nutrients for a particular pollutant removal in the myco-capsules of the present invention provide ease of formulation as well ease of use of the myco-capsules for a specific pollutant. The provision of barrier in the myco-capsule allows interaction of mycogranules and nutrients only when the capsule is added to wastewater. This mechanism makes mycocapsules self-sufficient as no more nutrient addition is required during wastewater treatment. The viability and dissolution/ dispersion time for both myco-capsules and myco-tablets was tested to show the difference in efficiency of pollutant removal for both the formulations. It was found that myco-capsules of the fungal strains have higher biomass production compared to the myco-tablet formulation of the same fungal strain over a period of one year of storage of the formulation at the temperature in the range of 25-30°C. Table 1 summarizes advantages of myco-capsules over myco-tablets. The products were also testified for the removal of metal-complex dye (Acid Blue 161), disazo pyrazolone pigment (Pigment Orange 34) and heavy metals [Cu (II) and Cr (VI)]. Hence, the present invention provides a simpler and low cost formulation of fungal strains i.e. myco-
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capsules with multiple metal and dye removal efficacy and such myco-capsules have been shown to be instrumental in simplifying the bioremediation of complex waste water.
Table 1: Advantages of myco-capsules over myco-tablets
Parameter
Mycotablet
Mycocapsule
Production
Production requires essential equipment (die, Automated Press Machine) and careful control of tableting process conditions
Production does not require these equipment or careful control of process conditions.
Viability
Loss in viability during tableting if conditions are not strictly met
Loss in viability is minimal
Contamina-tion
Chances of contamination due to unsterilized working conditions
Contamination is prevented as the process can be done in sterilized environment and post production the mycogranules are protected due to the encasing
User-friendly coding
Coding for different organisms and applications is difficult
The mycocapsules can be color coded for different organism and application for e.g. metal removal, dye removal or metal-dye mix removal
Efficacy
The efficacy of dye removal and metal removal (tested in this application) is lower as compared to mycocapsules especially after 6 months
The efficacy of dye/metal removal using a particular mycocapsule is higher than corresponding mycotablet. For eg. Myco-tablet (MT2-AT) after 12 months storage removed 83% Cr (VI) as compared to 99.3% removal with Mycocapsule (MC2-AT2).
Accordingly, the present invention provides a myco-capsule for bioremediation of waste water comprising:
a first part comprising a carrier based formulation of a fungal strain;
a second part comprising media specific for the fungal strain; and
a barrier for separating the first part and the second part,
wherein said first part is fitted into said second part and the barrier is inserted between the first part and the second part to separate the fungal strain and the media in the myco-capsule.
In an embodiment of the present invention, the fungal strain is selected from the group consisting of Aspergillus spp., Rhizopus spp., Beauveria spp., and a combination thereof.
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In another embodiment of the present invention, the Aspergillus spp. is Aspergillus lentulus or Aspergillus terreus, the Rhizopus spp. is Rhizopus oryzae, and Beauveria sp. is Beauveria bassiana.
In another embodiment of the present invention, the carrier is an organic base substrate; said organic base substrate is selected from the group consisting of Maize flour (MF), Rice flour (RF), Wheat flour (WF), and an organic base substrate having C/N ratio in the range of 23-32.
In yet another embodiment of the present invention, the myco-capsule is a pre-fabricated capsule having the first part and the second part; wherein said first part is smaller than the second part and the first part fits easily into the second part.
In yet another embodiment of the present invention, the pre-fabricated capsule is made up of gelatin.
In yet another embodiment of the present invention, the barrier used comprises gelatin.
In yet another embodiment of the present invention, the waste water comprises heavy metals or dyes or a combination thereof.
In yet another embodiment of the present invention, the carrier based formulation of the fungal strain is a powdered formulation; said powdered formulation is obtained by inoculating and incubating a fungal strain on the carrier.
In yet another embodiment of the present invention, the myco-capsule is color coded for a specific metal removal, specific dye removal or both metal-dye removal.
In yet another embodiment of the present invention, the myco-capsules comprise fungal strain in the range of 6.8 x 109 to 4.76 x 1010 CFU per capsule.
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In still another embodiment of the present invention, the ratio of the powdered formulation of fungal strain in the first part and the media in the second part is in the range of 0.1-0.3:0.6-0.4 on dry weight basis.
Still another embodiment of the present invention provides a method of preparation of myco-capsule comprising:
a. inoculating and incubating a fungal strain on a carrier to obtain a powdered formulation of the fungal strain;
b. filling the powdered formulation of the fungal strain in a first part of a pre-fabricated capsule;
c. filing a media specific for the fungal strain in a second part of the capsule; and
d. joining the first part with the second part and inserting a barrier to separate the first part and the second part to obtain the myco-capsule.
In yet another embodiment, the present invention provides a method in which the carrier is an organic base substrate selected from the group consisting of Maize flour (MF), Rice flour (RF) ,Wheat flour (WF), and an organic base substrate having C/N ratio in the range of 23-32.
In yet another embodiment, the present invention provides a method in which the fungal strain is selected from the group consisting of Aspergillus spp., Rhizopus spp., Beauveria spp., and a combination thereof.
In yet another embodiment, the present invention provides a method in which the Aspergillus spp. is Aspergillus lentulus or Aspergillus terreus, the Rhizopus sp. is Rhizopus oryzae, and Beauveria sp is Beauveria bassiana.
In yet another embodiment, the present invention provides a method in which ratio of the powdered formulation of fungal strain and the carrier in the myco-capsules is in the range of 0.1-0.3:0.6-0.4 on dry weight basis.
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In yet another embodiment, the present invention provides a method in which the myco-capsules comprise fungal strain in the range of 6.8 x 109 to 4.76 x 1010 CFU per capsule.
In yet another embodiment, the present invention provides a method in which ratio of the fungal strain and the carrier to obtain the powdered formulation of fungal strain is in the range of 4.6 x 109 to 6.8 x 109 CFU of fungal strain per 0.1g of the carrier.
In yet another embodiment, the present invention provides a method in which the barrier comprises gelatin.
In yet another embodiment, the present invention provides a method in which the pre-fabricated capsule used is made of gelatin.
In yet another embodiment, the present invention provides a method in which the myco-capsule is color coded for a specific metal removal, specific dye removal or both metal-dye removal.
In a still another embodiment, the present invention provides a method for bioremediation of waste water comprising adding myco-capsules into waste water; said myco-capsule comprising a fungal stain specific for a specific contaminant in the waste water; a media specific for the fungal strain, and a barrier separating the media and the fungal strain in the myco-capsule, and said waste water comprising heavy metals or dyes or a combination thereof; wherein dissolution of the capsule and the barrier takes place after addition in waste water thereby releasing the fungal strain and the media from the myco-capsules into the waste water; said media providing the required nutrition for the fungal strain to carry out remediation of the waste water.
EXAMPLES:
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present disclosure. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the subject matter.
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EXAMPLE 1: DEVELOPMENT OF MYCO-CAPSULES AND EVALUATION OF SHELF LIFE/ACTIVITY IN EXTERNAL MEDIA
The myco-granules of Aspergillus lentulus (accession no: FJI72995), Aspergillus terreus (accession no: KC354516) and Rhizopus oryzae (accession no: KC354517) (A. lentulus, A. terreus and R. oryzae, respectively, hereinafter) were developed by immobilizing the spores of the fungal strain on an organic base as disclosed previously (in Indian Patent Application no. 2590/DEL/2012). Briefly, each of the fungal strain was inoculated and incubated on a carrier which is an organic base substrate selected from the group consisting of Maize flour (MF), Rice flour (RF) and Wheat flour (WF). Using the earlier disclosed method, the fungal strain were propagated on the rice flour as organic base substrate (moisture content 60%) at 30 °C for 96 h. After 96 h contents of the flasks were taken out with the help of a spatula and dried at 45 °C for overnight. The dried mass was grinded and screened through sieve (pore size 105 μm) and again dried at 45 °C for 2 h. The powdered formulation thus obtained was called myco-granules. Such myco-granules of fungal strains were used to develop an encapsulated formulation called myco-capsules.
Precisely, 0.1 g of myco-granules of each fungal strain were filled into standard gelatin capsules (Gelatin ~ 80% to 85%, Moisture ~ 15, Other excipients ~ 0% to 5%; Size of capsule used:“00”) under sterilized conditions in a biosafety cabinet. The myco-capsules were stored at 30°C for a period of 12 months. At an interval of 3 months, the myco-capsules were tested for viability and metal/dye removal efficiency. One myco-capsule each was added to composite media (glucose 10.0 g L-1, yeast extract 2.5 g L-1, NH4NO3 0.5 g L-1, MgSO4.7H2O 0.1 g L-1, K2HPO4 0.5 g L-1, NaCl 1.0 g L-1, pH 6.5±0.2) containing metal [Cu(II) or Cr (VI)]and dye [Acid Blue 161 or Pigment Orange 34](initial concentration: 100 mg L-1) and incubated at 30ºC at 150 rpm for 120 h.
After the said duration, resultant biomass was measured by filtering the contents of the flask through Whatman No. 1 filter paper and estimating the dry weight of the biomass. The residual Cr (VI) concentrations in the filtrate was determined spectrophotometrically (PerkinElmer Lambda 35, UV Visible Spectrophotometer) at 540 nm using hexavalent chromium specific colorimetric DPC reagent. Residual Cu (II) concentration was quantified using atomic absorption
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spectrophotometer (Perkin Elmer AA200) after 120 h. Acid Blue 161 and Pigment Orange 34 concentrations were measured using UV-Visible spectrophotometer at the absorbance maxima (λmax) of respective dye (Acid Blue 161: 602 nm; Pigment Orange 34: 490 nm).
Figure 1 shows change in biomass production and dye removal in presence of external composite media by myco-capsules of A. lentulus, A. terreus and R. oryzae in presence of Acid Blue 161 and Pigment Orange 34, over a 12 month storage period. The viability of A. terreus myco-capsules was least affected in presence of both the dyes till 9 months of storage while A. lentulus myco-capsules were most sensitive. However, both A. terreus and A. lentulus myco-capsules retained more than 90% Acid Blue 161 dye decolorization activity after 9 months of storage. R. oryzae myco-capsules that showed 91% decolorization for Acid Blue 161 (Fig 1 (a)) at one month, declined to 76% after 9 months of storage. On the other hand, for Pigment Orange 34 dye (Figure 1 (b)), R. oryzae myco-capsules showed best performance with 100% Pigment Orange 34 removal after one month and more than 90 % removal after 9 months of storage. Interestingly, A. terreus myco-capsules displayed relatively poor performance for Pigment Orange 34 dye. The above results clearly indicate that myco-capsules are viable option for remediation of dyes and the choice of an appropriate organism for this formulation can give best results.
Figure 2 shows change in biomass production and metal removal in presence of external composite media by myco-capsules of A. lentulus, A. terreus and R. oryzae in presence of Cu (II) (Figure 2(a)) and Cr (VI) (Figure 2(b)), over a 12 month storage period. In spite of some changes in viability, the Cu (II) removal efficiency of A. terreus (74-63%) and A. lentulus (73-61%) myco-capsules was quite stable over a period of 12 months storage. R. oryzae myco-capsules had poor performance for metal removal including both Cu (II) and Cr (VI). On the other hand, Myco-capsules of A. terreus exhibited the best efficiency in terms of Cr (VI) removal where negligible decrease (0.02%) was observed after the end of 12 months storage. These results again established the selective nature of developed myco-capsules which can be exploited for Industrial applications. On the other hand, the results also indicate a possibility of getting better bioremediation kinetics by a use of mixtures or consortium of myco-capsules.
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EXAMPLE 2: MYCO-CAPSULES CONTAINING MULTIPLE OF STRAINS FOR METAL REMOVAL WITH EXTERNAL MEDIA
The performance of individual myco-capsule of three strains in removing test metal Cr (VI) was compared to that of myco-capsules having all the three fungal strains in the form of consortia. The myco-capsules having all the three fungal strains was prepared by adding myco-granules of A. lentulus, A. terreus and R. oryzae in 1:1:1 ratio (0.03:0.03:0.03 g) ratio as mentioned under Example 1. Then the myco-granules of each of the strain (0.1g each) were filled into the standard gelatin capsules (Size of capsule used: “00”) under sterilized conditions in a biosafety cabinet. Thereafter, myco-capsules of each of the three fungal strains and the myco-capsule containing three strains were inoculated in composite growth media (as used in Example 1) amended with metal (initial concentration 100 mg L-1). After inoculation, flasks were incubated at 30 °C and 150 rpm. Samples were intermittently withdrawn and analyzed for residual metal concentration till 120 h.
Figure 3 shows the performance of myco-capsules containing consortium of three strains over individual myco-capsules in terms of Cr (VI) removal with 100 mg L-1 initial concentration. The fungal consortium was able to remove chromium at a higher rate as compared to those achieved by A. lentulus and R. oryzae at a particular time. Although complete Cr (VI) removal was achieved by A. terreus, yet removal rate was higher with consortium as compared to the individual strains. No time was required for acclimatization towards metal resulting in increased metal removal rate with consortium. The results showed that consortium accounted for the complete removal of Cr (VI) in 72 h, while individual strain took longer time to remove Cr (VI).
EXAMPLE 3: PREPARATION OF PARTITIONED MYCO-CAPSULE HAVING INHERENT MEDIA
Application of myco-capsules in the above examples required the input of external media for supporting the growth of fungal strains in bioactive myco-granules for the dye/metal removal activity. To simplify the process and to make the myco-caspsules as complete package, partitioning of the myco-capsule was successfully attempted. Accordingly, a capsule having two parts was used for encapsulation of myco-granules for preparing a partitioned capsule having a barrier separating media and the bioactive myco-granules. The first part of the capsule is smaller
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in size as compared to the second part. Both first and second parts can fit into each other very easily. The smaller part was filled with the myco-granules (0.1g) of fungal strain (6.8 x 109 CFU). The larger part of the capsule was used to fill the particular nutrient media (0.6g) in powdered form The media composition used as external as well as inherent media was same and is: (glucose 10.0 g L-1, yeast extract 2.5 g L-1, NH4NO3 0.5 g L-1, MgSO4.7H2O 0.1 g L-1, K2HPO4 0.5 g L-1, NaCl 1.0 g L-1, pH 6.5±0.2). The ratio of the myco-granules in the first part and the media in the second part of the capsule is 0.1:0.6. The smaller part of the capsule was separated by the larger part by inserting a barrier in the capsule. The barrier used is water soluble in nature and is made up of gelatin. This barrier has its own significance. With the help of this barrier, mixing of myco-granules and media, which can result in vegetative growth and spoilage of the myco-granules, is inhibited inside the capsule. Further, the barrier helps in development of complete package as no external media is required to be added during the remediation process. The metal removal efficiency of partitioned myco-capsule with inherent media was tested by adding only the capsule to synthetic wastewater containing 100 mg L-1 Cu (II) and efficacy compared with the performance of non-partitioned capsule added into wastewater amended with external media and 100 mg L-1 Cu (II).
Figure 4 illustrate the metal removal efficiency of myco-capsules studied via two different strategies i.e. with external media and with inherent media in myco-capsules in the synthetic waste water with 100mg/l Cu (II) concentration. In first case, the composite media (CM1) was added externally to the waste water and myco-capsule containing myo-granules were added thereafter. And in other case, composite media (CM2) was encapsulated within the capsule along with the myco-granules, separated via water soluble membrane. It was observed that metal removal efficiency of the myco-capsule containing external media was slightly higher (3.3%) as compared to the myco-capsule with inherent media. However, the reduction in metal removal was not significantly higher compared to myco-capsule with inherent media. Hence, myco-capsule with inherent media can provide better option for the remediation process as it reduces the complexity of the ingredients to be added during treatment process.
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EXAMPLE 4: COLOR CODING OF PARTITIONED MYCO-CAPSULES FOR SPECIFIC APPLICATION
User-friendly color coding of myco-capsules was attempted for demarcating specific capsule for specific application i.e. metal removal, dye removal or metal-dye mix removal. The nutrient media to be added in the partitioned myco-capsule was customized using the optimized media for each application. Myco-capsules (partitioned having a barrier) were prepared using the process of Example 3 and using pre-fabricated colored gelatin capsules of white and pink color. White capsules contained media optimized (M1) for metal removal (Glucose 10 g L-1; Yeast extract 2 g L-1; Urea 0.7 g L-1; Ammonium nitrate 0.7 g L-1; Di-potassium hydrogen phosphate 0.5 g L-1; Magnesium sulphate 0.5 g L-1; Sodium chloride 1 g L-1 ). Pink capsules contained the media optimized (M2) for dye removal (Glucose 5 g L-1; Urea 0.3 g L-1; Ammonium chloride 0.2 g L-1; Di-potassium hydrogen phosphate 0.5 g L-1; Magnesium sulphate 0.5 g L-1; Sodium chloride 1 g L-1). The third type of capsule with composite media (CM) (glucose 10.0 g L-1, yeast extract 2.5 g L-1, NH4NO3 0.5 g L-1, MgSO4.7H2O 0.1 g L-1, K2HPO4 0.5 g L-1, NaCl 1.0 g L-1, pH 6.5±0.2) targeted for metal-dye mixture was designated with dual color (pink and white). The efficacy of white, pink and white-pink capsule was tested by adding each capsule separately into sterilized synthetic wastewater having metal (initial concentration: 100 mg L-1), Acid Blue 161 dye (initial concentration: 100 mg L-1) and mixture of both (50 mg L-1 each). The incubation was done at 30ºC at 150 rpm for 120 h followed by analysis of the residual pollutants.
Figure 5 indicates the removal efficiency of differently colour coded A.lentulus myco-capsule for different pollutant as well as for dye metal mixture. It is clear that pink capsules targeted for dye removal performed the best (98% removal) for Acid Blue 161 removal while showing relatively poor performance (93.20%) for Cr (VI) removal. On the other hand white capsules targeted for metal removal testified best (100% removal) for Cr (VI) removal and relatively poor (91%) for Acid Blue 161. Further, the white-pink capsules targeted for dye-metal mix pollutants showed good performance for the mixture with Cr (VI) 100% removal and Acid Blue 161 with 87% removal.
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EXAMPLE 5: COMPARISON BETWEEN MYCO-CAPSULES VS MYCOTABLETS
(a) Viability Comparison:
Viability of the myco-capsules and myco-tablets was evaluated in term of biomass produced by the product stored at 30ºC, over the year. The biomass produced by the myco-capsules (after every 3 months) was compared with the biomass produced by the myco-tablet in presence of Cr(VI) at 100 mg L-1 initial concentration kept at 30ºC at 150 rpm for 120 h. Biomass was filtered after 120h and kept in oven for 24h at 60ºC and weighed.
Figure 6 represents the viability comparison between the myco-capsules and myco-tablets in term of biomass produced by this formulation stored for 12 months at 30ºC, in presence of Cr (VI) at 100 mg L-1 initial concentration. It was observed that more biomass was produced by the myco-capsules of A. lentulus and A. terreus as compared to their myco-tablet formulation. Maximum decrease in the biomass production after 12 months was observed with A. lentulus myco-tablet (58.76%) as compared to the myco-capsules (47.09%). Similarly, with A. terreus, decrease in biomass with myco-tablets was 47.28% as compared to 46.05% for myco-capsules. These results clearly indicate that viability loss was more in case of myco-tablets as compared to myco-capsules, which might be due to the pressure applied during the tablet formation.
(b) Pollutant removal efficiency
Figure 7 clearly shows that myco-capsules produce higher Acid Blue 161 dye removal (82%) as compared to myco-tablets (69.5%) after 12 months of storage. Also, significantly higher Cr (VI) removal is shown by myco-capsules as compared to mycotablets after 12 months of storage. Better efficacy of myco-capsules is directly related to higher viability as discussed above. Hence, myco-capsules offer a better product for bioremediation of pure and mixed pollutants.
(c) Dissolution Time
Dissolution test was done for both the myco-tablet and myco-capsule. Tablet or capsule was added to 100 ml of water and time required for complete dispersion was measured. To carry out the uniformity of dispersion, three randomly selected tablets/ capsules from each formulation were placed in 100 ml of water and stirred gently until completely dispersed. Table 2 illustrates the results of dispersion test conducted on myco-tablet and myco-capsules.
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Table 2. Dissolution time of myco-capsules and myco-tablets of different fungal strains
Dissolution time (min)
A. lentulus
A. terreus
R. oryzae
Tablet
11.13±0.50
4.02±0.70
0.94±0.50
Capsules
1.15±0.40
1.15±0.50
1.15±0.60
It was found that complete dissolution of capsule wall and release of granulated formulation into the water takes around 1.15 min which is a considerably less time as compared to the myco-tablets (0.94 - 11.13 min). This is a significant advantage that can reduce the treatment time for pollutant water.
ADVANTAGES OF THE INVENTION
The myco-capsules as disclosed in the present application can be produced without a need for production equipment such as die, Automated Press Machine and careful control of tableting process conditions. Loss in viability is minimal during formulation of myco-capsules and thus, efficacy of dye/metal removal using a particular mycocapsule is higher than corresponding mycotablet. Further, contamination is prevented in myco-capsules as the process can be done in sterilized environment and post production the mycogranules are protected due to the encasing of the gelatin shell. Moreover, the myco-capsules can be color coded and optimized for different organisms and therefore, can be used for specific application for e.g. metal removal, dye removal or metal-dye mix removal.
Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible. As such, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiment contained therein.
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PRIOR ART REFERENCES:
1. Lamar, R. T., Dietrich, D. M., & Glaser, J. A. (1995). U.S. Patent No. 5,476,788. Washington, DC: U.S. Patent and Trademark Office.
2. Aust, S. D., Bumpus, J. A., & Tien, M. (1990). U.S. Patent No. 4,891,320. Washington, DC: U.S. Patent and Trademark Office.
3. Pereira, R. M., & Roberts, D. W. (1990). Dry mycelium preparations of entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana. Journal of Invertebrate Pathology, 56(1), 39-46.
4. Batta, Y. A. (2003). Production and testing of novel formulations of the entomopathogenic fungus Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes). Crop Protection, 22(2), 415-422.
5. Connick, W. J., Boyette, C. D., & Mcalpine, J. R. (1991). Formulation of mycoherbicides using a pasta-like process. Biological Control, 1(4), 281-287.
6. Biosigma S.A., (2014). Patent No. WO2014057443 A2.
7. Lakshmanan, V. I., and McCready, R. G. L. (1989). U.S. Patent No. US5084389 A.
8. Chidambaram, D., Liu, Y., & Rafailovich, M. H. (2013). U.S. Patent No. 8,367,109 B2. Washington, DC: U.S. Patent and Trademark Office.
9. Carpenter, R., Showell, M. S., Barnes, J., & Pal, N. (2014). U.S. Patent Application US20140342437 A1.
10. Bowers, R. C. (1982). Commercialization of microbial biological control agents.
11. Booth, S. R., & SHANKS JR, C. H. (1998). Potential of a dried rice/mycelium formulation of entomopathogenic fungi to suppress subterranean pests in small fruits. Biocontrol Science and Technology, 8(2), 197-206.
12. Gao, L., Sun, M. H., Liu, X. Z., & Che, Y. S. (2007). Effects of carbon concentration and carbon to nitrogen ratio on the growth and sporulation of several biocontrol fungi. Mycological research, 111(1), 87-92.
13. Khan, M. R., Majid, S., Mohidin, F. A., & Khan, N. (2011). A new bioprocess to produce low cost powder formulations of biocontrol bacteria and fungi to control fusarial wilt and root-knot nematode of pulses. Biological Control, 59(2), 130-140.
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14. Abraham, J., & Silambarasan, S. (2014). Biomineralization and formulation of endosulfan degrading bacterial and fungal consortiums. Pesticide biochemistry and physiology, 116, 24-31.
15. Stirling, G. R., Licastro, K. A., West, L. M., & Smith, L. J. (1998). Development of commercially acceptable formulations of the nematophagous fungus Verticillium chlamydosporium. Biological Control, 11(3), 217-223.
16. Connick, W. J., Boyette, C. D., & Mcalpine, J. R. (1991). Formulation of mycoherbicides using a pasta-like process. Biological Control, 1(4), 281-287.
17. Malik, A., Kaushik, P., & Mishra, A. (2012). Method for preparation of mycotablets for bioremediation and mycotablets thereof. Indian Patent Application no. 2590/DEL/2012.
We claim:
1. A myco-capsule for bioremediation of waste water comprising:
a first part comprising a carrier based formulation of a fungal strain;
a second part comprising media specific for the fungal strain; and
a barrier for separating the first part and the second part,
wherein said first part is fitted into said second part and the barrier is inserted between the first part and the second part to separate the fungal strain and the media in the myco-capsule.
2. The myco-capsule as claimed in claim 1, wherein the fungal strain is selected from the group consisting of Aspergillus spp., Rhizopus spp., Beauveria spp., and a combination thereof.
3. The myco-capsule as claimed in claim 2, wherein the Aspergillus spp. is Aspergillus lentulus or Aspergillus terreus, the Rhizopus spp. is Rhizopus oryzae, and Beauveria spp. is. Beauveria bassiana
4. The myco-capsule as claimed in claim 1, wherein the carrier is an organic base substrate; said organic base substrate is selected from the group consisting of Maize flour (MF), Rice flour (RF), Wheat flour (WF), and an organic base substrate having C/N ratio in the range of 23-32.
5. The myco-capsule as claimed in claim 1, wherein the myco-capsule is a pre-fabricated capsule having the first part and the second part; wherein said first part is smaller than the second part and the first part fits into the second part.
6. The myco-capsule as claimed in claim 5, wherein the capsule is made up of gelatin.
7. The myco-capsule as claimed in claim 1, wherein the barrier comprises gelatin.
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8. The myco-capsule as claimed in claim 1, wherein the waste water comprises heavy metals or dyes or a combination thereof.
9. The myco-capsule as claimed in claim 1, wherein the carrier based formulation of the fungal strain is a powdered formulation; said powdered formulation obtained by inoculating and incubating a fungal strain on the carrier.
10. The myco-capsule as claimed in claim 1, wherein the myco-capsule is color coded for a specific metal removal, specific dye removal or both metal-dye removal.
11. The myco-capsule as claimed in claim 1, wherein the myco-capsule comprises fungal strain in the range of 6.8 x 109 to 4.76 x 1010 CFU.
12. The myco-capsule as claimed in claim 1, wherein ratio of the powdered formulation of fungal strain in the first part and the media in the second part is in the range of 0.1-0.3:0.6-0.4 on dry weight basis.
13. A method of preparation of myco-capsule comprising:
a. inoculating and incubating a fungal strain on a carrier to obtain a powdered formulation of the fungal strain;
b. filling the powdered formulation of the fungal strain in a first part of a pre-fabricated capsule;
c. filing a media specific for the fungal strain in a second part of the capsule; and
d. joining the first part with the second part and inserting a barrier to separate the first part and the second part to obtain the myco-capsule.
14. The method of preparation of myco-capsule as claimed in claim 13, wherein the carrier is an organic base substrate selected from the group consisting of Maize flour (MF), Rice flour (RF), Wheat flour (WF), and an organic base substrate having C/N ratio in the range of 23-32.
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15. The method of preparation of myco-capsule as claimed in claim 13, wherein the fungal strain is selected from the group consisting of Aspergillus spp., Rhizopus spp., Beauveria spp., and a combination thereof.
16. The method of preparation of myco-capsule as claimed in claim 15, wherein the Aspergillus spp. is Aspergillus lentulus or Aspergillus terreus, the Rhizopus spp. is Rhizopus oryzae, and Beauveria spp. is. Beauveria bassiana
17. The method of preparation of myco-capsule as claimed in claim 13, wherein ratio of the fungal strain and the carrier to obtain the powdered formulation is in the range of 4.6 x 109 to 6.8 x 109 CFU of fungal strain per 0.1g of the carrier.
18. The method of preparation of myco-capsule as claimed in claim 13, wherein ratio of the powdered formulation in the first part and the media in the second part of the capsule is in the range of 0.1-0.3:0.6-0.4 on dry weight basis.
19. The method of preparation of myco-capsule as claimed in claim 13, wherein the barrier comprises gelatin.
20. The method of preparation of myco-capsule as claimed in claim 13, wherein the pre-fabricated capsule is made of gelatin.
21. The method of preparation of myco-capsule as claimed in claim 13, wherein the myco-capsule is color coded for a specific metal removal, specific dye removal or both metal-dye removal.
22. A method for bioremediation of waste water comprising adding myco-capsules into waste water; said myco-capsule comprising a fungal stain specific for a specific contaminant in the waste water; a media specific for the fungal strain, and a barrier
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separating the media and the fungal strain in the myco-capsule, and said waste water comprising heavy metals or dyes or a combination thereof; wherein dissolution of the capsule and the barrier takes place after addition in waste water thereby releasing the fungal strain and the media from the myco-capsules into the waste water; said media providing the required nutrition for the fungal strain to carry out remediation of the waste water.
