|Title of Invention||
" AN IMPROVED PROCESS FOR THE MINERALIZATION OF ALPHA HEXACHLOROCYCLOHEXANE USEFUL FOR TREATMENT OF INDUSTRY EFFLUENT AND BIOREMEDIATION OF CONTAMINATED SOIL"
|Abstract||The invention relates to an improved process for the mineralization of alpha hexachlorocyclohexane useful for treatment of industry effluent and bioremediation of contaminated soil. The process uses novel microbial consortium that can tolerate and effectively degrade higher concentration of α-HCH effluents. The process step involves treating HCH effluents or contaminated soil with microbial consortium consists of eight strains of different pseudomonas sp. and a strain of Fusarium sp. at a pH in the range of 4 to 10 and temperature 5 to 40°C to obtain α -HCH free material.|
|Full Text||This invention relates to an improved process for the mineralization of alpha hexachlorocyclohexane useful for treatment of industry effluent and bioreme-diation of contaminated soil.
In the process of the present invention potent microbial consortium that can degrade high concentration of alpha- hexachlorocyclohexane (HCH) was developed by us. Alpha-HCH is the major component (60-70% w/w) of commercially available technical HCH (commonly known as BHC or gammaxane), though this isomer has no insecticidal activity. (The insecticidal component of technical-HCH is the gamma-isomer known as Lindane). Alpha- HCH is highly persistent in the environment and is a major pollutant. However, "partial degradation of this compound to various intermediary metabolite in soil, particularly under anaerobic condition has been reported (Deo,P.G., Karanth,N.G. and Karanth,N.G.K, 1994. Crit. Rev. Microbiol. 20: 57-78). There are but a few reports on the aerobic biodegrada-tion of alpha-HCH. ( Kunhi,A.A.M. 1995, In R.Sankaran and K.S.Manja (eds) Microbes for Better Living., Proc. 35th Ann.Conf. AMI (Nov.1994) Conf. Secretariat, D.F.R.L., Mysore). A Psuedomonas sp. (Sahu,K.S., Pat-naik,K.K., Sharmila,M. and Sethunathan,N. 1990, Appl. Environ. Microbiol. 56: 3620- 3622.) and a Sphingomonas paucimobilis (Bhuyan,S., Sreedharan,B., Adhya,T.K. and
Sethunathan, N. 1993, Pestic. Sci. 38: 49-55) were shown to degrade alpha-HCH but at low concentrations (up to 11 ug/ml). Hence, it is imperative to isolate microbial strains or consortia that can degrade higher concentration of alpha-HCH, for developing process for treatment of industry effluents as well as for bioremediation of contaminated soil.
The main drawback of the hitherto known microbial strains is that they cannot degrade high concentration of the substrate and hence cannot be used for the treatment of industry effluents for eliminating the HCH residues that will be present in the effluent.
The main object of the present invention is to provide an improved process for the mineralization of alpha hexachlorocyclohexane useful for treatment of industry effluent and bioremediation of contaminated soil which obviates the drawbacks of the hitherto known methods.
Another object of the improved process of the present invention is to use microbial consortium that can tolerate and effectively degrade higher concentration of alpha -HCH for eliminating alpha-HCH from HCH-industry effluents and for bioremediation of contaminated soil.
Accordingly the present invention provides an improved process for the mineralization of alpha hexachlorocyclohexane useful for treatment of industry effluent and bioremediation of contaminated soil which comprises; contacting materials such as herein described containing alpha hexa chlorocyclohexane with a microbial consortium consists of eight
strains of different pseudomonas sp. and a strain of Fusarium sp. as herein described at a pH in the range of 4 to 10, at a temperature in the range of 5 to 40°C to obtain a -HCH free material.
A potent microbial consortium; coded CFR2001 was developed by us and acclimatized to increasing concentration of alpha-HCH (up to 100 ug/ml). The microbial mixed culture (Consortium CFR 2001) consisted of eight bacterial strains (different species of Pseudomonas) and one fungla strain (Fusarium sp.). This consortium could completely mineralise 100 ug/ml alpha-HCH within 72 hrs which is nearly 10 times higher than reported value. The consortium actively degraded alpha-HCH at a wide range of pH (4.0 -10.0) and temperature (5-40°C). This consortium can be effectively deployed for treatment of HCH- industry effluents and other materials polluted with alpha- HCH.
A novel enrichment technique was developed by which a potent alpha-HCH degrading consortium was
developed. In this new technique, samples from HCH-contaminated soil and sewage were enriched by long term enrichment in a semi continuous charcoal-packed column reactor filled with mineral salts medium with benzene, phenol and acetone (50 ul/L each) to start with- Once various microorganisms got established in the column it was fed with mineral salt medium containing technical-HCH (5ug/ml). The concentration of the technical- HCH was gradually increased to lOug/ml, while the concentrations of benzene, phenol and acetone were gradually decreased and finally withdrawn. Broth samples were collected every week and further enriched in shake flasks in mineral salts medium containing lOug/ml of alpha-HCH as the sole source of carbon. Samples drawn from the column reactor after 6 months and further enriched in shake flasks for 8 weeks showed utilization of alpha-HCH.
The chemicals used in the media and reagents were of analytical grade.
The basal mineral medium used for enrichment and
growth of CFR 2001 contained ( per litre of distilled
water) 0.675 g KH PO ; 5.455 g Na HPO ; 0.25g NH NO ;
2 4 2 4 4 3
0.2g MgSO .7H O; 0.1g Ca(NO ) and 1 ml of trace miner-
4 2 3 2
al solution containing (mg/ml) FeSO .7H 0, 1.0;
MnSO .H O, 1.0; CuCl .2H O, 0.25; Na MoO .2H O, 0.25;
4 2 2 2 2 4 2
and H BO , 0.10. The pH of the medium was 7.5. Re-3 3
quired amount of alpha-HCH was added tp the medium as
described later. For studying the effect of pH on
alpha-HCH degradation, the required pH was adjusted by
altering the buffer salt concentrations or by the
addition of acid (HNO ) or alkali (NaOH) solutions.
The Luria agar medium used for plating the microbial consortium to study the taxonomy of the bacterial strains contained (per litre of distilled water) lOg tryptone; 5.Og yeast extract, 6.0g NaCl and 20.0 g of agar agar (pH 7.0).
The cultivation of the consortium in shake flasks
was carried out in 50 ml of mineral medium containing
required amount of growth substrate (alpha-HC) taken in
o 250 ml Erlenmeyer flasks and incubating at 30 C on a
rotary shaker (150 rpm). For maintaining the consortium, mineral agar plates/slopes containing technical HCH
(25 ug/ml) were used and the consortium was grown for
one week at 30 C and then preserved at 4 C. Alternatively, liquid culture grown on mineral salts medium
containing 25 ug/ml alpha-HCH for 24 h was stored at
o 4 C.
The growth of the consortium was determined by estimating the total protein in the biomass by a modified method of Lowry (Lowry,O.H., Rosebrough,N.J., Farr,A.L. and Randall,R.J. 1951, J. Biol. Chem. 193: 265-275) as follows: The cells were harvested, washed with distilled water and suspended in 3.4 ml of distilled water. 0.6 ml of 20% NaOH was added, mixed and digested in a constant boiling water bath for 5 min. Total protein in cooled sample was determined by Lowry's method using Folin Ciocalteau reagent.
Quantitative estimation of chloride was done by mercury
(II) thiocyanate method (Bergmann,J.G. and Sanik,Jr.J.
1957. Anal. Chem., 29: 241-243). To 2 ml of aliquot
of the culture supernatant, 0.2 ml of 0.25M ammonium
iron (III) sulphate [Fe(NH (SO ) .12H O] in 9M nitric
4 4 2 2
acid was added followed by the addition of 0.2 ml of a saturated solution of mercury (II) thiocyanate in ethanol. Absorbance of the sample solution was measured at 460 nm by a spectrophotometer (Shimadzu UV, 160A, Japan). The amount of chloride was computed using chloride standard curve. Quantitative determination of the substrate residue (alpha-HCH) was done by TLC. The culture broth was extracted with thrice its volume of a mixture of acetone: hexane (1:8) for 15
min. by thorough mixing. The solvent layer was pooled, evaporated and taken in convenient volume of hexane. Known volume was loaded onto silica gel G coated plate (0.8 mm) and developed in cyclohexane. The plate is air dried and alpha-HCH was detected by spraying the plate with 2% solution of a o- tolidin in acetone. The spots were delineated by marking with a needle and the area was measured. The concentration was computed from a standard plot of log concentration Vs. square root of the area, prepared for pure alpha-HCH.
The alpha-HCH degrading microbial consortium was developed in the laboratory by a novel enrichment tech nique. The isolation technique involved a two step enrichment; first in a semi-continuous chemostat and the in shake flasks as follows: cylindrical glass column chemostat (5x50 cm) was filled with wood charcoal cubes (1-2 cm) and basal mineral medium (per
litre, 0.675g, KH PO ; 5.455g Na HPO ; 0.25g of NH NO ;
2 4 2 4 4 3
0.2g MgSO , 7H 0; O.lg Ca(N0 ) and 1 ml of trace
4 2 3 2
minerals solution (in mg/ml FeSO .7H 0, 1.0; MnSO .H O,
4 2 4 2
1.0; NaMoO , 0.25; H BO , 0.1 and CuCl .2H O, 0.25)
4 3 3 2 2
containing acetone, benzene and phenol as carbon
sources (each 50 ul/1 (v/v). The pH of the medium was
7.5. To this was added aqueous suspension of soil samples collected from sugarcane fields, paddy fields, contaminated with HCH and sewage samples, which were filtered through a clean cheese cloth. The column reactor was maintained semi-continuously, by withdrawal and recirculation of the medium 3-4 times per day and partial replacement of the medium"once in 2-3 days. After two weeks the concentrations of acetone, benzene and phenol were gradually reduced and started feeding mineral medium containing technical HCH (5 ug/ml), the concentrations of which was gradually increased and brought to 10 ug/ml after 4 weeks. This semi-continuous operation was continued for a total time of 6 months, during which period broth samples were collected and further enriched in shake flasks.
For shake flask enrichment the same medium containing alpha-HCH was used as a sole source of carbon and energy. 50 ml medium taken in 250 ml Erlenmeyer flasks to which the required amount of alpha-HCH was
added as acetone solution (50 ul) were incubated at
o 30 C on a Emenvee rotary shaker (150 rpm). Shake flask
enriched consortium after 8 weeks (with weekly transfers to fresh medium) showed utilization of alpha-HCH
as determined by the release of stoichiometric amounts of chloride.
The alpha-HCH degrading consortium CFR 2001 obtained above was further acclimatized to higher concentrations of alpha-HCH from 10 ul/ml upto 100 ug/ml . Alpha-HCH in these cases was dissolved in acetone and added to dry sterile flask. The flasks were kept openin a laminar hood for 15 min to allow complete evaporation of acetone. Mineral medium was then added to the flask and inoculated with the consortium and incubated on a rotary shaker (150 rpm) at 30 Deg C. Samples were analyzed periodically for release of chloride. The consortium which could degrade 10 ug/ml alpha-HCH completely was used for studies with 25 ug/ml alpha-HCH which was then carried for the degradation of 50 ug/ml and finally 100 ug/ml alpha-HCH. Complete degradation of each concentration of alpha-HCH was obtained by repeated transfers to fresh medium containing same concentrations of alpha-HCH. (Degradation was checked by the estimation of chloride released as well as by the complete disappearance of alpha-HCH). By this method, the rate of degradation of alpha-HCH significantly improved. The total time of about 15 days taken
for complete mineralisation of 100 ppm of alpha-HCH was reduced to 3 days after acclimatization. The consortium which could degrade. 100 ug/ml alpha-HCH was plated on Luria agar medium to find out the types of bacteria/fungi involved in the degradation. It showed eight different bacterial strains and a fungal strain. The bacteria were identified as different species of Pseudomonas according to Bergey's Manual of Determinative Bacteriology (Table-1). The fungus was identified as Fusarium sp.
Table-1: The list of microorganisms present in the alpha-HCH degrading consortium CFR 2001
Microorganisms 1. Bacteria
Pseudomonas fluorescens CFR1002 P.stutzeri CFR1003 P.pseudoflava CFR1004 P.palleronii CFR1005 P.diminuta CFR1006 P.mendocina CFR1007 P. caryophilli CFR1008 P. solanacearum CFR1009 Fungus Fusarium sp. CFR217
The following examples are given by way of illustration that should not construed the limit of the scope of the present invention. Example-1 To test the ability of alpha-HCH degrading consortium to completely mineralise alpha-HCH, the following experiments were done. The mineral medium having
0.675g KH PO ; 5.455g Na HPO ; 0.25g NH NO ; 0.2g
2 4 2 4 4 3
MgSO .7H 0; O.lg Ca(NO ) and 1 ml of trace mineral
4 2 3 2
solution per litre was used. (Trace mineral solution
had FeSO .7H 0, l.Omg; MnSO .H O; 1.0. mg; NaMoO ,
4 2 4 2 4
0.25mg; H BO , 0.1 mg and CuCl .2H 0 0.25 mg per ml).
3 3 2 2
Alpha-HCH was added at 25 ppm (25 ug/ml). 25 ml of
medium was taken in 250 ml conical flasks in tripli
cate. The medium was inoculated with the cells of the
consortium so as to obtain an initial protein content
of 3 2 ug protein per ml. The flasks were incubated on
o a rotary shaker at 150 rpm at 30 C. Samples were drawn
at regular intervals of 3 h and analyzed for growth (total biomass protein) inorganic chloride and residual substrate. Almost complete disappearance of the substrate was observed after 48 h of incubation and also 100% chloride release was observed at 48 h of incubation indicating complete mineralisation of the compound .
Degradation of 50 ppm (50 ug/ml) of alpha-HCH by alpha-HCH degrading consortium CFR 2001 was carried out in shake flasks. The composition of the medium except concentration of the substrate and all other conditions were exactly the same as in Example-1..
50 ug.ml of alpha-HCH was degraded completely within 7 2 h of incubation with stoichiometric release of chloride. The substrate also disappeared completely at 72h growth period as determined by TLC.
Example-3 The ability of alpha-HCH degrading consortium CFR 2001 in degrading 100 ul/ml of alpha-HCH was studied. Except for substrate (alpha-HCH) concentration, -medium and other culture conditions were similar to that of example-1. The consor tium was found to degrade 100 ppm (100 ug/ml) of alpha-HCH in 72 hr incubation period.
The ability of the alpha-HCH degrading consortium CFR 2001 to degrade 10 ug/ml of alpha-HCH at different media pH was tested by varying the pH from 3.0 to 11.0. The pH of the medium was varied by changing the concentration of buffering salts KH PO and Na HPO and also
2 4 2 4
by adjusting with acid (HNO ) and alkali (NaOH) solu-
tions. Inoculated flasks were incubated for 24 hr at
o 30 C on a rotary shaker (150 rpm). Except for the pH
and the substrate concentrations, all other conditions were the same as in Example-1. Growth was determined as total biomass protein and inorganic chloride and residue was estimated.
The consortium showed ability to grow at a wide range of pH from 4.0 to 10.0, the best HCH-degrading ability in terms of release of Cl being at pH ranging from 6.5 8.0.
The ability of alpha-HCH degrading consortium CFR
2001 to degrade alpha-HCH at different temperatures
was tested by growing the cultures at 4, 8, 20, 25, 30,
o 3 5 and 40 C under stationary conditions for 48 h.
Medium used was same as in Example-1 with 10 ug/ml
alpha-HCH. Although growth was observed at a wide
o range of temperatures from 2 0 to 40 C, optimum growth
and degradation of alpha-HCH in terms of Cl release
o was observed at 30 C.
The ability of the consortium to degrade alpha-HCH
at a fairly wide range of temperatures particularly at
o cold conditions (temperature as low as 5 C) is an
advantage for its use in treatment technologies.
1. An improved process for the mineralization of alpha hexachlorocyclohexane useful for treatment of industry effluent and bioremediation of contaminated soil which comprises; contacting materials such as herein described containing alpha hexa chlorocyclohexane with a microbial consortium consists of eight strains of different pseudomonas sp. and a strain of Fusarium sp. as herein described at a pH in the range of 4 to 10, at a temperature in the range of 5 to 40°C to obtain a -HCH free material.
2. An improved process for the mineralization of alpha hexachlorocyclohexane useful for treatment of industry effluent and bioremediation of contaminated soil substantially as herein described with reference to the examples.
|Indian Patent Application Number||2448/DEL/1995|
|PG Journal Number||46/2012|
|Date of Filing||29-Dec-1995|
|Name of Patentee||COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH|
|Applicant Address||RAFI MARG NEW DELHI-110001, INDIA.|
|PCT International Classification Number||C12P 1/00|
|PCT International Application Number||N/A|
|PCT International Filing date|