Title of Invention

METHOD FOR BIOLOGICAL TREATMENT OF AQUEOUS EFFLUENTS CONTAINING NON AQUEOUS PHASE LIQUID POLLUTANTS

Abstract

The invention relates to a method of biological treatment of aqueous effluents containing non-aqueous phase liquid pollutants, the method comprising contacting the aqueous effluents with a consortium of oil tolerant algae and oil degrading bacteria, the algae being responsible for maintaining a near neutral pH, immobilizing the bacteria and facilitating the settlement of suspended biomass. The invention also relates to a consortium of immobilized algae and bacteria for biological treatment of aqueous effluents containing non-aqueous phase liquid pollutants, the consortium comprising of oil tolerant algae and oil degrading bacteria, the algae being responsible for maintaining a near neutral pH, immobilizing the bacteria and facilitating the settlement of suspended biomass.

Full Text

FORM 2 THE PATENTS ACT, 1970 (39 of 1970) As amended by the Patents (Amendment) Act, 2005 & The Patents Rules, 2003 As amended by the Patents (Amendment) Rules, 2006 COMPLETE SPECIFICATION (See section 10 and rule 13) TITLE OF THE INVENTION Biological treatment of aqueous effluents containing Non Aqueous Phase Liquid pollutants APPLICANTS Name : Indian Institute of Technology, Bombay Nationality : an autonomous research and educational institution established in India by a special Act of the Parliament of the Republic of India under the Institutes of Technology Act 1961 Address : Powai, Mumbai 400076, Maharashtra, India INVENTORS Names : Suparna Mukherji and Anal Chavan Nationality : both Indian,Nationals Address : both of Indian Institute of Technology, Bombay, Centre for Environmental Science and Engineering, Powai, Mumbai 400076, Maharashtra, India PREAMBLE TO THE DESCRIPTION The following specification particularly describes the nature of this invention and the manner in which it is to be performed: Field of Invention The present invention relates to a method of biological treatment of aqueous effluents containing non-aqueous phase liquid pollutants using the consortium of algal and bacterial cultures. The invention also relates to a consortium of algal and bacterial cultures for biological treatment of aqueous effluents containing non-aqueous phase liquid (NAPL) pollutants. The term non-aqueous phase liquids used in this specification refers to oil / hydrocarbons that are often present in wastes from petrochemical industries, petroleum refineries or motor oil reforming industries. Background of invention Conventionally, treatment of aqueous effluents containing non-aqueous phase liquid pollutants is carried out using API separator, tilted plate separator and dissolved air floatation units followed by suspended growth biological treatment processes, such as, the activated sludge process. US 5527974 describes separating natural fats and oils from glycerol water by a combination of phase separation and microfiltration process. The dissolved air flotation process used for reducing oil concentration before biological treatment generates large volumes of oily sludge that requires treatment prior to disposal. The activated sludge process is energy intensive due to the requirement of aerators during treatment. Moreover, biological treatment of hydrocarbon rich wastewater using activated sludge process is associated with the following problems: (1) Effluents having high COD/BOD ratio cannot be treated (2) Settleability of the sludge is poor due to low F/M (Food to microorganism) ratio (3) During the treatment, extracellular polymers consisting of lipids, proteins and carbohydrates are often produced which adversely affects sludge settling (4) Biological inhibition occurs due to toxic compounds in the aqueous effluents, which necessitates very long sludge retention times and long periods of acclimation or start-up and (5) It produces a large amount of sludge. Biological treatment in a rotating biological contactor (RBC) overcomes the above mentioned problems. Moreover, the rotating discs ensure sufficient oxygen transfer in the system eliminating the need for separate aerators for oxygen supply.- However, atteihpts to successfully attach and grow 2 stable biofilms of oil degrading bacteria on the rotating discs of the reactor have so far not proved successful. This is because the bacteria alone does not form a stable biofilm on the discs. The treatment of aqueous effluents containing non-aqueous phase liquid pollutants, such as oils, using oil degrading bacteria generally results in a reduction in pH due to formation of fatty acids produced during oil degradation. Acidic conditions are not conducive to bacterial growth. Another problem associated with biological treatment using bacterial cultures is the poor settleability of sludge containing the oil degrading bacteria. US 4348285 and US 4432869 disclose methods for treatment of liquid agricultural wastes using a combination of algal^and bacterial cultures. US 6033559 discloses significant enhancement in degradation /mineralization of n-hexadecane and chrysene by a constructed microbial«mat (comprising Oscillatoria and purple autotrophic bacteria in a laminated structure annealed tightly together by slimy secretion from various microbial components) in the presence of inorganic- support materials, such as, clay / zeolite / bentonite. However, algal-cultures typically have low tolerance to non-aqueous phase liquids, especially at high concentrations. Detailed description of invention Accordingly, the present invention provides a consortium of algal and bacterial cultures for biological treatment of aqueous effluents containing non-aqueous phase liquid pollutants, the consortium comprising oil degrading bacteria immobilized on oil acclimatized algae, the algae being responsible for maintaining a near neutral pH, immobilizing the bacteria and facilitating the settlement of suspended biomass. In one embodiment, the invention provides a consortium of oil acclimatized algal and bacterial cultures, the acclimatization being carried out for a period of around 30 days and upto a concentration of 0.6 % (v/v) of the non-aqueous phase liquid. In another embodiment, the invention provides a method of biological treatment of aqueous effluents containing non-aqueous phase liquid pollutants, the method comprising 3 contacting the aqueous effluents with a consortium of oil degrading bacteria and oil acclimatized algae, the algae being responsible for maintaining a near neutral pH, immobilizing the bacteria and facilitating settlement of the suspended biomass. In a further embodiment, the invention provides a method of carrying out biological treatment of aqueous effluents containing non-aqueous phase liquid pollutants, in a rotating biological contactor, the method comprising contacting the aqueous effluents with an immobilized biofilm comprised of a consortium of oil degrading bacteria and oil acclimatized algae formed on the discs of the reactor, the algae being responsible for maintaining a near neutral pH, immobilizing the bacteria and facilitating settlement of the suspended biomass. The rotating biological contactor (RBC) used is known as a typical experimental set-up for attached growth biological treatment. The RBC used as stated in the invention consisted of a semi-circular trough with three stages. It was provided with 27 discs of 14 mm diameter. The total effective surface area of the discs was 0.83 m , surface area of the tank was 0.09 m2 and the working volume of the' reactor was 4 litres. The trough was of stainless steel so as to reduce adsorption of oil and the discs were made of acrylic. The acrylic discs acted as a substratum for growth of the algal-bacterial consortium. They were mounted on a 0.6 m horizontal PVC shaft of 0?025 m diameter. The thickness of the acrylic discs was 3 mm and spacing'between the discs was 14 mm. The discs were 35 % submerged in the trough and were rotated at 10 rpm using a 0.25 HP motor and reduction gear system. The discs were rotated at a peripheral velocity of 0.7 m/min so as to allow sufficient oxygen transfer in the system. Sampling ports were provided at the end of each stage for effluent collection. Three ports were provided at the bottom of each stage for cleaning and maintenance of the reactor. The trough was mounted on a frame made up of mild steel angle sections. One 100 watt bulb was mounted on an angle iron frame to give an average light intensity of about 1000-1100 lux at the surface of the RBC. A timer arrangement was provided so as to maintain light: dark cycle of 18:6 for the growth of the algal culture. To separate the biomass from the treated effluent, a stainless steel effluent tank with sampling port and a stirring arrangement was provided. Sampling of the 4 effluent was conducted after proper stirring to Obtain a representative sample. The aqueous effluents used had a Total Petroleum Hydrocarbon (TPH) loading not greater than 24 g TPH/m2 .d and a COD loading not greater than 63 g COD/m2.d. The pH of the aqueous effluent was maintained within a range of 7.4 to 7.9 by the use of the consortium of algal and bacterial cultures. The aqueous effluents had a concentration of non-aqueous phase liquids up to 0.6 % (v/v) comprising largely of straight chain aliphatic compounds. The consortium of algal and bacterial cultures of the present invention is characterized by a synergistic association. In addition to providing a support for the oil degrading bacteria, the algae are autotrophic and provide oxygen for aerobic degradation of the non- aqueous phase liquids. This enhances the degradation efficiency of the non-aqueous phase liquids. Moreover, the algae provides a buffered environment that will resist the lowering of pH during the biological treatment thereby providing a conducive environment for the survival of the oil degrading bacteria The bacterial culture of the present invention was isolated from oil contaminated sediments of the Arabian Sea obtained from the vicinity of an oil field in the Bombay High Region and was identified as Burkholderia cepacia (99.6% match in 16S rDNA analysis with B. cepacia LMG 12614t2, a strain available in Belgian Coordinated Collections of Microorganisms (BCCM), Gent, Belgium). This culture of B. cepacia (designated as strain IITB-SM-1) was submitted, to Institute of Microbial Technology (IMTECH, Chandigarh, India). The accession number allotted is MTCC 5332. The algal culture was obtained from the surface of rocks near Powai lake at IIT Bombay, India. Microscopic examination was carried out to observe the morphology of the algal culture present in the biofilm of the reactor. The algal culture was predominantly composed of blue-green algae (cyanobacteria) that could grow in the presence of cycloheximide. The presence of blue-green algal culture was also • confirmed by the presence of phycobiliprotein pigments which are absent in other algal cultures. Some filamentous green algal culture was also present and its growth was inhibited in the presence of cycloheximide. The blue-green, algal culture was predominantly composed of Phormidium. In microscopic observation Phormidium was characterized by its distinct 5 filaments, trichomes with false branching and without heterocysts, absence of spores and filaments forming a thallus with more or less confluent sheath. The other species of blue-green algal culture present included Oscillatoria and Chroococcus. Oscillatoria was characterized by its distinct filaments, trichomes with false branching, without heterocysts, absence of spores, clearly visible cells in the trichome, more or less straight trichomes not spirally coiled and absence of sheath. Chroococcus was characterized by its unicellular / united colonies with colorless individual sheath, and by the non-vesicular and spherical cells. In a typical experiment, the feed containing non-aqueous phase liquids was treated with a consortium of algal and bacterial cultures in a rotating biological contactor. Prior to treatment, a biofilm of the consortium of algae and bacteria was developed over the discs of the reactor. Initially, mass cultivation of fresh water algal culture capable of tolerating high concentration of diesel oil and B. cepacia capable of degrading diesel oil was performed. The algal culture was incubated at room temperature without shaking under illumination of 1100 lux, with light: dark cycle of 18:6. The B. cepacia culture was incubated at 28 ± 2°C at 120 rpm in a rotary shaker. The reactor was operated with diesel in the oily waste serving as the only source of carbon and energy. Nutrient formulation for good growth of both the microorganisms determined through batch studies was as follows (concentration in mg/1): di-sodium EDTA:(0.5), KH2P04 (8.5), K2HP04.3H20 (21.8), Na2HP04.7H20 (33.4), NaN03 (750), MgSG4.7H20 (370), CaCl2.2H20 (7), KCl (500), citric acid (3), Na2C03 (20)-and ferric ammonium citrate (3) and trace element solution containing H3BO3 (0.062), MnCl2.4H20 (0.2807), ZnCl2 (0.1363), CuCl2.2H20 (0.0392), Na2Mo04.2H20 (0.0254), CoCl2.6H20 (0.0382), NiCl2 (0.013), FeCl2.4H20 (0.7016) and Na2S04 (0.142). The RBC was first operated in the batch mode to facilitate the growth of both bacterial as well as algal cultures on the rotating discs of the RBC. The RBC trough was filled with 3 litres of algal culture and 1 litre of bacterial culture. Since the algal culture demonstrated a tendency for forming clumps, vortexing with glass beads was employed for obtaining a uniform suspension. The concentration of algal culture and bacterial culture, in terms of 6 VSS (Volatile Suspended Solids) was 780 and 220 mg/1, respectively. The algae to bacteria ratio in the trough was 3.55:1 on VSS basis and 3:1 on volume basis. During this time, the diesel concentration in the reactor was maintained at 0.2%. It was observed that about 0.8-1 litre of water evaporated daily. To maintain constant water level in the reactor, fresh nutrient media was replaced daily and 1.6-2 ml diesel was added, assuming a similar rate of diesel evaporation. The development of algal-bacterial biofilm was observed after 17 days of inoculation. After the development of algal-bacterial biofilm on the discs of RBC, diesel concentration in the feed was gradually increased. During this phase of operation, the reactor was operated in semi-batch mode. The semi-batch mode of operation was started with 0.2% diesel. Everyday, the reactor contents was drained out completely, stirred on a magnetic stirrer and the content was analyzed. The reactor was operated with 0.2% diesel for a period of 10 days. Subsequently, for the next 10 days, i.e., from 10th day to 19th day, the reactor was operated with 0.4% diesel. Subsequently, from 20th to 29th day, the reactor was operated with 0.6% diesel. Thus, the algal-bacterial consortium was gradually acclimatized to 0.6 % diesel in 30 days. During the treatment, the rotating biological contactor was operated in a continuous flow-through mode. Separate pumping systems (using peristaltic pumps), was used for inflow of nutrients and substrate, i.e., diesel. The reactor was operated with 0.6% diesel using a hydraulic retention time of 24 hrs. The corresponding hydraulic loading, TPH (Total Petroleum Hydrocarbon) loading and COD (Chemical oxygen demand)loading were 0.0048 m3/m2.d, 23.91 g TPH/m2.d and 62.65 g COD/m2.d, respectively. Throughout, the dissolved oxygen level in the effluent was in the range of 4-5 mg/1. The growth of bacterial culture was predominant in the first stage of the reactor, due to greater amount of substrate availability and gradually decreased over the second and third stages. In contrast, growth of the algal culture was minimum in the first stage of the reactor and maximum in the third stage of the reactbr. The thickness of the biofilm was found to be highest in the first stage of the reactor and least in the third stage of the 7 reactor. Total biomass (algae + bacteria) in the first stage, second stage and third stage varied from 45-55, 30.3-46.4, 14.8-34.4 mg/cm2, respectively. Based on chlorophyll estimation, algal biomass in the first, second and third stage were 0.6-4.0, 2.5-6.5, 5-11 mg/cm , respectively. The ratio of total volatile solids (TVS) to total solids (TS) per unit area of the RBC disc defined as Disk biomass index (DBI) [(gTVS/cm2) / (gTS/cm2)] was always found to be greater than 0.95, in all the stages. For total biomass exceeding 55 mg/cm2, severe sloughing of the biofilm was observed. The biofilm thickness was controlled by rotating the discs periodically for a short time at an increased velocity to remove excessive growth. To avoid clogging in the system, the biomass collected at the bottom of the trough was drained after a one month interval. Each disc on a rotating shaft contained attached biomass (algae and bacteria) that reacted with the surrounding oily wastewater in the trough. The greater the wastewater flow or concentration of oil, the greater the number of discs that were required for producing the same degree of treatment. Also, the greater the degree of treatment required, the greater the number of discs that were required for providing the same quality of treated wastewater. Characteristics of the aqueous effluent before and after treatment are summarized in Table 1. Table. 1 Characteristics of aqueous effluent before and after treatment Parameters Concentrations before treatment Concentrations after treatment PH 7.4-7.6 7.6-7.9 Alkalinity (as CaC03) 50-60 450-550 TPH 4961.4 31-35 COD 13,000* 400-450 N03"-N 35-40 15-16 PO/'-P 6-7 8-9 TSS 2-3 70-80 AH concentrations are expressed in mg/1, except pH. *: COD based on theoretical calculation; TPH is Total Petroleum Hydrocarbons; COD is Chemical Oxygen Demand; TSS is Total Suspended Solids It can be seen from the table (Table. 1) that there is substantial reduction of TPH and COD of the aqueous effluents due to biological treatment in the reactor. It was found that the system was capable of treating about 99.31% TPH and 96.8% COD, with residual TPH and COD of 31-35 mg/1 and 400-450 mg/1, respectively. Effluent total suspended 8 solids (TSS) concentration was found to be 70-80 mg/1. Nitrate nitrogen and phosphate phosphorus in the effluent was found to be in the range of 15-16 mg/1 and 8-9 mg/1, respectively. It can also be seen that no considerable change in pH and no considerable increase in TSS occurred in the liquid after treatment. This is attributed to the buffering activity and the enhanced settleability of the sludge due to the presence of the algae. Alkalinity in terms of CaCC>3 concentration increased due to photosynthetic activity of the algae. Although the invention has been described in detail in the foregoing section for the purpose of illustration, it is to be understood that sucsh detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims. 9 We Claim 1. A method of biological treatment of aqueous effluents containing non-aqueous phase liquid pollutants, the method comprising contacting the aqueous effluents with a consortium of oil acclimatized algae and oil degrading bacteria, the algae being responsible for maintaining a near neutral pH, immobilizing the bacteria and facilitating the settlement of suspended biomass. 2. A method of carrying out biological treatment of aqueous effluents containing non-aqueous phase liquid; pollutants, in a rotating biological contactor, the method comprising contacting the aqueous effluents with an immobilized biofilm comprising a consortium of oil acclimatized algae and oil degrading bacteria, the algae being responsible for maintaining a near neutral pH, immobilizing the bacteria and facilitating the settlement of suspended biomass. 3. The method as claimed in claims 1 or 2 wherein the aqueous effluents has a TPH loading not greater than 24 g TPH/m2 .d and a COD loading not greater than 63 g COD/m2.d. 4. The method as claimed in any one of the claims 1 to 3 wherein pH of the aqueous effluents is maintained within a range of 7.4 to 7.9. 5. The method as claimed in any one of the claims 1 to 3 wherein the non-aqueous phase liquid pollutants have a concentration of up to 0.6 % (v/v). 6. The method as claimed in any one of the claims 1 to 5 wherein the non-aqueous phase liquid pollutants comprises straight chain aliphatic compounds. 7. The method as claimed in any one of the claims 1 to 5 wherein the non-aqueous phase liquid pollutants comprises diesel oil. 8. A consortium of algal and bacterial cultures for biological treatment of aqueous effluents containing non-aqueous phase liquid pollutants, the consortium comprising oil degrading bacteria immobilized on oil acclimatized algae , the algae being responsible for maintaining a near neutral pH, immobilizing the bacteria and facilitating the settlement of suspended biomass. 10 9. The consortium as claimed in claim 8 wherein the algal and bacterial cultures are acclimatized for a period of up to 30 days and to a concentration of up to 0.6 % (v/v) of the non-aqueous phase liquid pollutants. 10. The consortium as claimed in claim 9 wherein the bacterial culture comprises Burkholderia cepacia. 11. The consortium as claimed in claim 9 wherein the algal culture comprises blue green algae 12. The consortium as claimed in claim 11 wherein the blue green algae comprises Phormidium, Oscillatoria and Chroococcus 13. The consortium as claimed in claim 12 wherein the blue green algae predominantly comprises Phormidium Dated this 28th day of December 2006 11 ABSTRACT Title: Biological treatment aqueous effluents containing Non Aqueous Phase Liquid pollutants The invention relates to a method of biological treatment of aqueous effluents containing non-aqueous phase liquid pollutants, the method comprising contacting the aqueous effluents with a consortium of oil tolerant algae and oil degrading bacteria, the algae being responsible for maintaining a near neutral pH, immobilizing the bacteria and facilitating the settlement of suspended biomass. The invention also relates to a consortium of immobilized algae and bacteria for biological treatment of aqueous effluents containing non-aqueous phase liquid pollutants, the consortium comprising of oil tolerant algae and oil degrading bacteria, the algae being responsible for maintaining a near neutral pH, immobilizing the bacteria and facilitating the settlement of suspended biomass.

Documents:

2177-MUM-2006-ABSTRACT(15-7-2009).pdf

2177-MUM-2006-ABSTRACT(23-3-2010).pdf

2177-mum-2006-abstract(granted)-(11-6-2010).pdf

2177-mum-2006-abstract.doc

2177-mum-2006-abstract.pdf

2177-MUM-2006-CANCELLED PAGES(23-3-2010).pdf

2177-MUM-2006-CLAIMS(15-7-2009).pdf

2177-MUM-2006-CLAIMS(AMENDED)-(23-3-2010).pdf

2177-mum-2006-claims(granted)-(11-6-2010).pdf

2177-mum-2006-claims.doc

2177-mum-2006-claims.pdf

2177-MUM-2006-CORRESPONDENCE(01-12-2009).pdf

2177-MUM-2006-CORRESPONDENCE(15-7-2009).pdf

2177-mum-2006-correspondence(23-3-2010).pdf

2177-MUM-2006-CORRESPONDENCE(25-08-2008).pdf

2177-MUM-2006-CORRESPONDENCE(3-3-2010).pdf

2177-MUM-2006-CORRESPONDENCE(6-3-2009).pdf

2177-mum-2006-correspondence(ipo)-(29-6-2010).pdf

2177-mum-2006-correspondence-received.pdf

2177-mum-2006-description (complete).pdf

2177-MUM-2006-DESCRIPTION(COMPLETE)-(15-7-2009).pdf

2177-mum-2006-description(granted)-(11-6-2010).pdf

2177-MUM-2006-FORM 1(15-7-2009).pdf

2177-MUM-2006-FORM 1(23-3-2010).pdf

2177-mum-2006-form 13(23-3-2010).pdf

2177-mum-2006-form 18(9-3-2007).pdf

2177-mum-2006-form 2(15-7-2009).pdf

2177-mum-2006-form 2(granted)-(11-6-2010).pdf

2177-MUM-2006-FORM 2(TITLE PAGE)-(15-7-2009).pdf

2177-MUM-2006-FORM 2(TITLE PAGE)-(23-3-2010).pdf

2177-mum-2006-form 2(title page)-(granted)-(11-6-2010).pdf

2177-mum-2006-form 26(13-2-2010).pdf

2177-mum-2006-form 8(29-5-2007).pdf

2177-mum-2006-form-1.pdf

2177-mum-2006-form-2.doc

2177-mum-2006-form-2.pdf

2177-mum-2006-form-3.pdf

2177-mum-2006-marked copy(23-3-2010).pdf

2177-MUM-2006-OTHER DOCUMENT(23-3-2010).pdf

2177-MUM-2006-REPLY TO EXAMINATION REPORT(23-3-2010).pdf

2177-MUM-2006-SPECIFICATION(AMENDED)-(23-3-2010).pdf