FORM 2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES, 2003
COMPLETE SPECIFICATION (See section 10, rule 13)
1. Title of the invention: A MICROBE MEDIATED PROCESS FOR WASTEWATER
TREATMENT AND USES THEREOF
2. Applicant(s)
NAME NATIONALITY ADDRESS
HINDUSTAN Indian Petroleum House 17, Jamshedji
PETROLEUM Tata Road Churchgate, Mumbai -
CORPORATION LIMITED 400020 Maharashtra, India
3. Preamble to the description
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it
is to be performed.

FIELD OF INVENTION
[001] The present disclosure relates to the field of wastewater treatm ent utilizing a
Candida strain HP-CAT01.
BACKGROUND OF THE INVENTION
[002] Wastewater is water contain ing solid suspended matter as well as other impurities generated as a byproduct of m unicipal, industrial or comm ercial activities. The impurities released in wastewater would vary depending on the industry concerned, for example, in a food a nd beverage industry; the impurities would range from particulate matter and surf actants to flavoring and coloring ag ents, fats etc. Similarly, in a petroleum refinery the wa stewater produced would be rich in hydrocarbon material as well as phenolics and sulphur rich com pounds. When such organic matter is releas ed into the envir onment, it gradually decays by a process o f oxidation. However, this utilizes the disso lved oxygen present in water bodies, thus endangering the survival of aquatic life. Therefore, the water needs to be treated before being discharged into the environment.
[003] Primary treatment of refinery wastewater is basically a gravity separation method that removes the bulk of oil and susp ended solids in the wastewater and thus reduces the load on downstream treatment methods. The secondary treatm ent of wastewater is usually a biol ogical process in which m icroorganisms are allowed to consume the organic matter to produce carbon dioxide and water, as well as the energy required for their growth and m ultiplication. Usually this is accomplished either by a trickling bed reactor or an activated sl udge process. In th e more commonly us ed activated sludge process, settled sewage and wastewater are mixed in an aeration tan k where dissolved oxygen is continuously bubbled so as to provide both aeration as well as a rap id mixing action. In su ch aerated conditions, microbes grow extremely well and absorb the organic m atter from the wa stewater/sludge thus reducing its organic content.

[004] However, the m icrobes used in the aerat ion tank could be very sensitive to certain toxic compounds that are present in the wastewater itself. For example, phenol concentrations greater than ~ 50 ppm are toxic to m icrobes as it can interfere with the integrity of the cell membrane. Similarly, chloride concentrations also need to be in the range of ~ 1000 ppm for a healthy aeration tank microbiota. But this can be difficult to achieve as changes in process operations of the industry concer ned or the use of seawater for cooling operations etc. can lead to higher chloride c oncentrations in the wastewater, which could adve rsely impact the microbes in th e aeration tank and even prove to be fatal. Theref ore, there is need for a process or an organism that is robust and can handle different concentrations of pollutants without any effect on its own viability and thus augment the performance of the aeration tank as a whole.
SUMMARY OF THE INVENTION
[005] In an aspect of the present disclosure, there is provided a method of wastewater treatment, said method comprising the steps: (a) obtaining an isolated Candida strain HP-CAT01 having MTCC accession num ber 25057; and (b) co ntacting said microorganism with wastewater, wherein sa id method results in reduction of phenol content of wastewater from about 20-200mg/L to about 0-4mg/L in 6 -10 hours. [006] In an aspect of the pres ent disclosure, there is provided an isolated Candida strain HP-CAT01 having MTCC accession number 25057 for use in treatment of wastewater to reduce phenolic content.
[007] These and other features, aspects, and advantages of the present subject m atter will be better understo od with reference to the following description and app ended claims. This summary is provided to introdu ce 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 lim it the scope of the claimed subject matter.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[008] The following drawings form part of the present specification and are included
to further illustrate asp ects of the presen t disclosure. The disclosure may be better
understood by reference to the drawings in co mbination with the detailed description
of the specific embodiments presented herein.
[009] Figure 1 depicts the phe nol tolerance of the Candida strain HP-CAT0 1, in
accordance with an embodiment of the present disclosure.
[0010] Figure 2 depicts the phenol consumption by the Candida strain HP-CAT01, in
accordance with an embodiment of the present disclosure.
[0011] Figure 3 depicts the chloride ion tolerance of the Candida strain HP-CAT01, in
accordance with an embodiment of the present disclosure.
[0012] Figure 4 depicts the sul phite tolerance of the Candida strain HP-CAT01, in
accordance with an embodiment of the present disclosure.
[0013] Figure 5 depicts the temperature tolerance of the Candida strain HP-CAT01, in
accordance with an embodiment of the present disclosure.
[0014] Figure 6 depicts the am monia tolerance of the Candida strain HP-CAT01, in
accordance with an embodiment of the present disclosure.
[0015] Figure 7 depicts the tolerance of Candida strain HP-CAT01 to phenol, chloride
ion, sulphite, ammonia, or combinations thereof, in accordance with an embodiment of
the present disclosure.
[0016] Figure 8 depicts the phenol degradation by Candida strain HP-CAT01 in the
presence of chloride, sulphite, amm onia, or combinations thereof, in accordance with
an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Those skilled in the art will be aware that the present disclosure is subject to variations and m odifications other than t hose specifically described. It is to be understood that the present disclosure include s all such variations and modifications. The disclosure also includes all su ch steps, features, compositions and compounds

referred to or indicated in this specif ication, individually or collectively, and any and all combinations of any or more of such steps or features.
Definitions
[0018] For convenience, before further descrip tion of the present disclosure, certain
terms employed in the spec ification, and exam ples are collected here. These
definitions should be read in the light of the rem ainder of the disclosu re and
understood as by a person of skill in the art. The terms used herein have the m eanings
recognized and known to those o f skill in th e art, however, for convenience an d
completeness, particular terms and their meanings are set forth below.
[0019] The articles “a”, “an” and “the” are used to refer to one or to m ore than one
(i.e., to at least one) of the grammatical object of the article.
[0020] The terms “comprise” and “comprising” are used in the inclusive, open sense,
meaning that additional elements may be included. It is not intended to be construed as
“consists of only”.
[0021] Throughout this specification, unless the context requires otherwise the word
“comprise”, and variations such as “comprises” and “comprising”, will be understood
to imply the inclusion of a stated elem ent or step or group of elem ent or steps but not
the exclusion of any other element or step or group of element or steps.
[0022] The term “including” is used to m ean “including but not lim ited to”.
“Including” and “including but not limited to” are used interchangeably.
[0023] Unless defined otherwise, all technical a nd scientific term s used herein have
the same meaning as commonly understood by one of ordinary skill in the art to which
this disclosure belongs. A lthough any methods and materials similar or equivalent to
those described herein can be used in th e practice or tes ting of the d isclosure, the
preferred methods, and materials are now described. All publications mentioned herein
are incorporated herein by reference.
[0024] The present disclosure is not to be lim ited in scope by the specif ic
embodiments described herein, which are intended for the purposes of exemplification

only. Functionally-equivalent products, com positions, and methods are clearly within the scope of the disclosure, as described herein.
[0025] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment, said m ethod comprising the steps: (a) obta ining an isolated Candida strain HP-CAT01 having MTCC accession number 25057; and (b) contacting said strain with wastewater, wherein said method results in reduction of phenol content of wastewater from about 20-200mg/L to about not detectable-4mg/L in 6-10 hours. [0026] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment, said method comprising the steps: (a) an isolated Candida strain HP-CAT01 having MTCC accession num ber 25057; and (b) co ntacting said microorganism with wastewater, wherein sa id method results in reduction of phenol content of wastewater from about 20-200m g/L to about no t detectable-4mg/L in 8 hours.
[0027] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment as described herein, wherein said m ethod results in reduction of Biological Oxygen Dem and of wastewater from about 150-200m g/L to about 10-20mg/L in 6-10 hours.
[0028] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment as described herein, wherein said m ethod results in reduction of Biological Oxygen Dem and of wastewater from about 150-200m g/L to about 10-20mg/L in 8 hours.
[0029] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment as described herein, wherein said m ethod results in reduction of Chemical Oxygen Demand of wastewater fr om about 300-600mg/L to 50-100mg/L in 6-10 hours.
[0030] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment as described herein, wherein said m ethod results in reduction of Chemical Oxygen Demand of wastewater fr om about 300-600mg/L to 50-100mg/L in 8 hours.

[0031] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as describ ed herein, wherein said wastew ater is refin ery
wastewater.
[0032] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said method is suitable to treat
wastewater having phenol concentration of up to 1600ppm.
[0033] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said method is suitable to treat
wastewater having chloride concentration of up to 20000ppm.
[0034] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said method is suitable to treat
wastewater having sulphite concentration of up to 300ppm.
[0035] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said method is suitable to treat
wastewater having ammonia concentration of up to 300ppm.
[0036] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said method is carried out at a
temperature of 25-45°C.
[0037] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said method is carried out at a
temperature of 30°C.
[0038] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said method is carried out at a
temperature of 37°C.
[0039] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said method is carried out at a
temperature of 42°C.

[0040] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as describ ed herein, wherein said strain can use phenol as a
carbon source.
[0041] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wh erein said strain can use phenol as a sole
carbon source.
[0042] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising phenol at a con centration of 25ppm as sole carbon source
is about 0.15.
[0043] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising phenol at a con centration of 50ppm as sole carbon source
is about 0.1.
[0044] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising phenol at a concentration of 100ppm as sole carbon source
is about 0.19.
[0045] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising phenol at a concentration of 200ppm as sole carbon source
is about 0.22.
[0046] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising phenol at a concentration of 400ppm as sole carbon source
is about 0.6.
[0047] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)

grown in media comprising phenol at a concentration of 800ppm as sole carbon source
is about 0.75.
[0048] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising phenol at a c oncentration of 1000ppm as sole carbon
source is about 0.9.
[0049] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising phenol at a c oncentration of 1200ppm as sole carbon
source is about 0.9.
[0050] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising phenol at a c oncentration of 1600ppm as sole carbon
source is about 0.4.
[0051] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 1000ppm is about 4.1.
[0052] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 2000ppm is about 3.8.
[0053] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 3000ppm is about 4.
[0054] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)

grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 4000ppm is about 5.8.
[0055] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 5000ppm is about 5.1.
[0056] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 6000ppm is about 5.3.
[0057] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 7000ppm is about 5.
[0058] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 8000ppm is about 3.9.
[0059] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 9000ppm is about 4.8.
[0060] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 10000ppm is about 3.5.
[0061] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)

grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 15000ppm is about 4.4.
[0062] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in m edia comprising glucose as sole carbon source, and chloride ion
concentration of 20000ppm is about 5.1.
[0063] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising glucose as sole carbon source, and sulphite concentration
of 25ppm is about 8.8.
[0064] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising glucose as sole carbon source, and sulphite concentration
of 50ppm is about 7.8.
[0065] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising glucose as sole carbon source, and sulphite concentration
of 75ppm is about 8.2.
[0066] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising glucose as sole carbon source, and sulphite concentration
of 100ppm is about 9.
[0067] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising glucose as sole carbon source, and sulphite concentration
of 125ppm is about 8.
[0068] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)

grown in media com prising glucose as sole carbon source, and sulphite concentration
of 150ppm is about 9.
[0069] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising glucose as sole carbon source, and sulphite concentration
of 175ppm is about 6.
[0070] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising glucose as sole carbon source, and sulphite concentration
of 200ppm is about 6.6.
[0071] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising glucose as sole carbon source, and sulphite concentration
of 250ppm is about 8.4.
[0072] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media com prising glucose as sole carbon source, and sulphite concentration
of 300ppm is about 7.8.
[0073] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said m ethod is capable of reducing
biological oxygen demand of wastewater by about 35-65%.
[0074] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said m ethod is capable of reducing
chemical oxygen demand of wastewater by about 60-75%.
[0075] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising glucose as sole carbon source, and ammonia concentration
of 25ppm is about 11.

[0076] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising glucose as sole carbon source, and ammonia concentration
of 25ppm is about 11.
[0077] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising glucose as sole carbon source, and ammonia concentration
of 50ppm is about 11.
[0078] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising glucose as sole carbon source, and ammonia concentration
of 75ppm is about 12.
[0079] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising glucose as sole carbon source, and ammonia concentration
of 100ppm is about 11.
[0080] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising glucose as sole carbon source, and ammonia concentration
of 125ppm is about 11.
[0081] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising glucose as sole carbon source, and ammonia concentration
of 150ppm is about 13.
[0082] In an em bodiment of t he present disc losure, there is provided a m ethod of
wastewater treatment as described herein, wherein said strain cell growth density (OD)
grown in media comprising glucose as sole carbon source, and ammonia concentration
of 200ppm is about 12.

[0083] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment as described herein, wherein said strain cell growth density (OD) grown in media comprising glucose as sole carbon source, and ammonia concentration of 250ppm is about 11.
[0084] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment as described herein, wherein said strain cell growth density (OD) grown in media comprising glucose as sole carbon source, and ammonia concentration of 300ppm is about 13.
[0085] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment as described herein, wherein said strain cell growth density (OD) grown in m edia comprising glucose as sole carbon source, and chloride ion concentration of up to 20000pm, is comparable to growth in absence of chloride ion. [0086] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment as described herein, wherein said strain cell growth density (OD) grown in media com prising glucose as sole carbon source, and sulphite concentration of up to 300pm, is comparable to growth in absence of sulphite. [0087] In an em bodiment of t he present disc losure, there is provided a m ethod of wastewater treatment as described herein, wherein said strain cell growth density (OD) grown in media comprising glucose as sole carbon source, and ammonia concentration of up to 300pm, is comparable to growth in absence of ammonia. [0088] In an em bodiment of the present disclo sure, there is pro vided an is olated Candida strain HP-CAT01 having MTCC accession number 25057 for use in treatment of wastewater to reduce phenolic content.
[0089] In an em bodiment of t he present disclo sure, there is provided a m ethod of wastewater treatment as described herein, wherein said method is suitable to treat wastewater having phenol concentration of up to 1600 ppm, chloride concentration of up to 20000 ppm, sulphite concentration of up to 300 ppm and ammonia concentration of up to 300 ppm.

[0090] Although the subject matter ha s been described in cons iderable detail with reference to certain preferred embodiments thereof, other embodiments are possible.
EXAMPLES
[0091] The disclosure will no w be illu strated with working ex amples, which is
intended to illustrate the working of disclosure and not intend ed to take restrictively to
imply any limitations on the scope of the present disclosure. Unless defined otherwise,
all technical and scientific term s used herein have the sam e meaning as commonly
understood to one of ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclo sed methods and composition s, the exem plary methods, devices and materials are described he rein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
Example 1
Hydrocarbon degradation profile of Candida HP-CAT01 strain
[0092] The primary treatment of wastewater in refineries aims to reduce the oil load
in the effluent treatment plant (ETP), but sometimes when the feed oil content is more,
due to any change in refinery operations or some malfunction in the primary treatment
itself, the o il content in the a eration tank cou ld become higher. Th is is of concern
because the oil will form a layer in the tank and prevent the atmospheric oxygen from
penetrating into the tank thus af fecting the viability of the microorganisms in it. So an
organism capable of degrading the oil content in wastewater is an added advantage. An
isolated Candida strain HP-CAT01 having MTCC accession num ber 25057, when
introduced into wastewater generated from an oil refinery, was able to degrade almost
51% of crude oil and 15% of residual o il, when grown in a minim al medium
containing just the oil as a carbon source. The results are summarized in the table given below.
[0093] Table 1

Degradation in minimal-oil medium in 8 days
Basra Crude oil 51.0 ± 9.6* %
Residual oil 15.8 ± 1.9* %
Control oil without strain 7.1 ± 5.1* %
*Results are average of triplicates
[0094] The phenol and hydrocarbon rich inlet of the aeration tank can be diverted into a fermenter where the strain is grow n in a continuous m anner (with a residence time of approxim ately 24 – 48 hours) and then fed to the aeration tank. (i.e. a fermenter has to be connected in between the hydrocarbon containing feed inlet and the aeration tank). This design enables both onsite cultivation of the biocatalyst as well as reduces the hydrocarbon load on the aeration tank
Example 2
Phenol degradation profile of Candida HP-CAT01 strain
[0095] Phenolic compounds are comm only generated during regular petroleum refinery operations and are ro utinely present in the ef fluent generated from refineries. Phenol is highly toxic d ue to its ac idic nature and ability to disrupt th e integrity of microbial cell membranes, because of which it is commonly used as a disinfectant. So, phenolic compounds entering the aeration tank of the efflue nt treatment plant (ET P) can be toxic to the m icrobes present. Although the microorganisms in aeration tanks are usually very robust a nd can tolerate a wide a rray of che micals, phenol concentrations above a certain threshold are lethal. Therefore, if the phenol content of the effluent stream increases, due to any routine change in refinery operation, the overall health of the ETP aeration tank can get compromised. An organism with a high phenol tolerance is thus an ideal candidate to be used in the ETP. [0096] The strain HP-CAT01 was grown in minimal media containing phenol (as the only carbon source) in concentrations ranging from 25 – 2000 ppm and it was observed that growth occurr ed even at levels as hi gh as 1600 ppm (Figure 1). The

effluent stream of refineries usually contains approximately 150 – 200 ppm of phenol, thus, from Figure 1; it is evident that the strain can withstand almost eight times the phenol content normally seen in effluents.
[0097] In addition, it is also important to degrade the phenol present in effluent streams so that it does not end up pollu ting water bodies. This strain could reduce ~1000 ppm phenol to less than 5 ppm (which is the acceptable norm for phenol in water as per the Central Pollution Control Bo ard) within 8 hours (Figure 2). The rapid phenol degradation and high tole rance of the strain m akes it an ideal m odification to wastewater treatment, as it helps to re duce the toxic effect of phenol on the other microbes in the aeration tank, thus improving the overall life of the ETP.
Example 3
Chloride tolerance of Candida strain HP-CAT01
[0098] Most refineries are located along the coast and it is a common practice to use seawater for cooling, cleaning and other operations. However, when this highly saline water reaches the aeration tank alo ng with the effluent stream, it can s everely inhibit the growth of m icrobes in the tank. Therefor e, it is im portant to use salt tolerant microbes in the aeration tank. The Candida HP-CAT01 strain was grown in m inimal media containing glucose with salt concentrations ranging from 0 – 20000 ppm and the cell growth was very good even at extrem ely high concentrations of 20000 ppm of sodium chloride (which is m uch higher than the concentration expected in sea water i.e. 10000 ppm) (Figure 3).
Example 4
Sulphite tolerance of Candida strain HP-CAT01
[0099] Refinery effluent streams generally contain ~ 30 – 80 ppm of sulphite but the HP-CAT01 strain can grow well at sulphite co ncentrations that are up to ten times higher (Figure 4).
Example 5
Reduction in Biological Oxygen Demand (BOD), and Chemical Oxygen Demand
(COD) of wastewater treated by Candida strain HP-CAT01

[00100] Biological oxygen dem and or BOD is a measure of the dissolved oxygen required in a water body by m icrobes to oxidize the dissolved organic matter within a specific period of tim e. It is quite sim ilar to COD or chemical oxygen dem and, the only difference being that the latter is more general and inc ludes the oxidation of all chemical content in water irrespective of its origin. Both BOD and COD are used to estimate the level of organic m atter pollution in water bodies and the levels of both these values have to be within permissible limits before the water treated in the ETP is discharged into the environm ent. Candida strain HP-CAT01 has the capacity to metabolize a wide array of chem icals because of which it can reduce the BOD by almost 35 – 65% and the COD by 60 – 75%, th us making it an attractive organism to be used in any kind of effluent treatment, particularly refinery wastewater treatment.
Example 6
Temperature tolerance of Candida strain HP-CAT01
[00101] The HP-CAT01 strain is able to grow well over a broad temperature range of 30 – 42 0C (Figure 5). This em phasizes the r obustness of the strain as it would continue to grow and multiply well even if the aeration tank temperature varies for any reason.
Example 7
Ammonia tolerance of Candida strain HP-CAT01
[00102] The HP-CAT01 stra in is also ab le to tolerate ammonia, which is u sed regularly in various refinery ope rations and that is considered to be lethal to the other organisms in the aeration tank in concentrations greater than 100 ppm. The HP-CAT01 strain could grow com fortably in m inimal media containing as high as 300 ppm ammonia (Figure 6), indicating its high robustness.
Example 8
Phenol degradation profile of Candida strain HP-CAT01 in presence of various
inhibitors
[00103] In addition to the to lerance of the strain for individual growth inhibitory factors like chlorides, sulphites, am monia, high temperature etc. th e ability of strain

HP-CAT01 to degrade phenolics in the presence of these moieties was also determined experimentally. Briefly, HP-CAT01 was grown in minimal media containing 400 ppm phenol as the sole carbon source in presen ce of com binations of inhibitors like chlorides (15000 ppm ), sulphites (100 ppm ) and amm onia (300 ppm). About 90% phenol degradation was observed in 24 ho urs, even in the pres ence of th ese compounds (Figure 7).
[00104] Further, the cell growth of the said strain is quite robust in the presence of multiple inhibitory factors such as chloride, or chlo ride and sulph ate (Figure 8). However, growth was retarded in the pr esence of chloride, sulphite, and ammoni a (Figure 8), even though as seen from Figure 7, the strain w as still able to substantially degrade phenol.
Example 9
Cost effective method of high cell density cultivation of Candida strain HP-CAT01
[00105] The minimal nutritional requirements and robustness of this strain enables its cultivation in cheap media like sugarcane juice or jaggery and minimal salts, instead of using complex and expensive components like yeast extract, tryptone, peptone etc. that are normal components of media used traditionally for yeast growth like Yeast Extract Peptone Dextrose Broth (YEPD), Terrific Brot h etc. Sugarcane juice or jaggery which usually contains 14 – 18% and 60 – 80% sucros e, respectively is diluted to achieve a final sugar concentration of 5% a nd mixed with minimal salts (MgSO4 – 0.5 g/L, KCl 0.5 g/L, KH2PO4 – 1 g/ L, NaNO3 – 2.5 g/L) and used to grow HP-CAT01 strain to high cell densities such as 50 – 70. In fact, this strain can also be cultivated in th e refinery effluent itself with m inimal salt supplementation as it contains sufficient amounts of carbon and nitrogen to support the growth, thus enabling the generation of huge amounts of bioinoculant using inexpensive and easily available components.” [00106] Overall, the present disclo sure provides an efficien t and superior m ethod of wastewater treatment for rem oval of pheno lics from wastewater. The m ethod of the instant disclosure is particularly robust as evidenced by its effectiveness in removal of phenolics in the presence of high concentrations of inhi bitory compounds such as

ammonia, chloride, and sulphites, wh ich are comm only found in wastewater, especially generated from refineries. This represents a cost effective and rob ust mechanism of treating wastewater.

I/We claim:
1. A method of wastewater treatment, said method comprising the steps:
a. obtaining an isolated Candida strain HP-CAT01 having MTCC
accession number 25057; and
b. contacting said strain with wastewater,
wherein said method results in reduction of phenol content of wastewater from about 20-200mg/L to about 0-4mg/L in 6-10 hours.
2. The method as claimed in claim 1, wherein said method results in reduction of Biological Oxygen Dem and of wastewater from about 150-200m g/L to about 10-20mg/L in 6-10 hours.
3. The method as claimed in claim 1, wherein said method results in reduction of Chemical Oxygen Demand of wastewater from about 300-600mg/L to 50-100mg/L in 6-10 hours.
4. The method as claimed in claim 1, wherein said method is suitable to treat wastewater having phenol concentration of up to 1600 ppm.
5. The method as claimed in claim 1, wherein said method is suitable to treat wastewater having chloride concentration of up to 20000 ppm.
6. The method as claimed in claim 1, wherein said method is suitable to treat wastewater having sulphite concentration of up to 300 ppm.
7. The method as claimed in claim 1, wherein said method is suitable to treat wastewater having ammonia concentration of up to 300 ppm.
8. The method as claim ed in claim 1, wher ein said method is carried out at a temperature of 25-45°C.
9. The method as claimed in claim 1, wherein said method is suitable to treat wastewater having phenol concentration of up to 1600 ppm, chloride concentration of up to 20000 ppm, sulphite concentration of up to 300 ppm and ammonia concentration of up to 300 ppm.
10. The method as claimed in claim 1, where said strain can be cultivated in a cost effective manner using wastew ater itself by diverting som e of the phenol and

hydrocarbon containing inlet of th e aeration tank to a reacto r) instead of usin g conventional media such as sugarcane juice, jaggery, etc.
11. An isolated Candida strain HP-CAT01 having MTCC accession number 25057 for use in treatment of wastewater to reduce phenolic content.