Process for bio-sludge reduction in hydrocarbon refinery effluent treatment plant through microbial interventions

Abstract

A process for treatment of hydrocarbon refinery wastewater producing a low bio-sludge, the process including utilizing microbial consortia comprising at least one species of Pseudomonas and at least one species of Bacillus in a ratio of 10:1 to 1:10. The species of Pseudomonas and species of Bacillus have constitutive expression of at least one hydrocarbon degrading gene. The species of Pseudomonas are selected from the group consisting of Pseudomonas stutzeri (MTCC 25027), Pseudomonas aeruginosa (MTCC 5389), Pseudomonas aeruginosa strain IOC DHT (MTCC, 5385), Pseudomonas putida IOCR1 (MTCC 5387), Pseudomonas putida IOC5a1 (MTCC 5388) and a mutant thereof. The species of Bacillus are selected from the group consisting of Bacillus subtilis (MTCC 25026), Bacillus substilis (MTCC 5386), Bacillus thermoleovorans (MTCC 25023), Bacillus stearothermophilus (MTCC 25030), Lysinibacillus sp. (MTCC 25029), Lysinibacillus sp. (MTCC 5666) and a mutant thereof. The microbial consortia is used in concentration of at least 10.sup.2 cfu/ml.

Claims

1. A process for hydrocarbon refinery wastewater treatment for producing low bio-sludge, the process comprising utilizing an immobilized microbial consortia comprising species of Pseudomonas and species of Bacillus in a ratio of 10:1 to 1:10, wherein the microbial consortia comprises Pseudomonas stutzeri (MTCC 25027), Bacillus subtilis (MTCC 25026), Lysinibacillus sp. (MTCC 5666), Pseudomonas putida IOCR1 (MTCC 5387) and Lysinibacillus sp. (MTCC 25029), and wherein the microbial consortia is used in concentration of at least 10.sup.2 cfu/ml.

2. The process as claimed in claim 1, wherein the treated hydrocarbon refinery wastewater comprises mixed liquor suspended solids (MLSS) ranging from 700-1300 ppm.

3. The process as claimed in claim 1, wherein the process is carried out at a temperature ranging from 5 C. to 50 C.

4. The process as claimed in claim 1, wherein the process is carried out at a pH ranging from 4.0 to 9.0.

5. The process as claimed in claim 1, wherein the microbial consortia expresses at least one hydrocarbon degrading gene selected from the group consisting of: Phosphopantetheinyl transferase (sfp, sfp0), Surfactin synthetase complex (srfAD), Rhamnosyltransferase subunit A (rh1A), Rhamnosyltransferase subunit B (rh1B), Lichenan-specific enzyme IIA component (licA), Lichenan-specific enzyme IIB component (licB), Lichenanspecific enzyme IIC component (licC), Catechol 1, 2 dioxygenase (CatA), Catechol dioxygenase (C12O, C23O), Catechol 2, 3 dioxygenase (xy1E), Naphthalene dioxygenase (nahH, nahI), Aromatic-ring hydroxylating dioxygenase (RHD), Alkane monooxygenase (AlkB, AlkB1), Alkane dioxygenase (nahAc), n-alkane monooxygenase (CYP153), Long chain alkane monooxygenase (ladA), Flavin-binding monooxygenase (almA), Sulfide: quinone oxidoreductase (sqr), Persulfide dioxygenase (pdo), Chromate Reductase (ChrR) or a combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a general scheme for treatment of hydrocarbon refinery wastewater using specific microbial consortia disclosed in present application.

DETAILED DESCRIPTION OF THE INVENTION

(2) While the invention is susceptible to various modifications and/or alternative processes and/or solvent system, specific embodiment thereof has been shown by way of examples and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular processes and/or temperature, pH, ratios, quantity and strains disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims.

(3) The figures and protocols have been represented where appropriate by conventional representations, showing only those specific details that are pertinent to understanding of the embodiments of the present invention and not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

(4) The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.

(5) Any particulars and all details set forth herein are used in the context of some embodiments and therefore should not be necessarily taken as limiting factors to the attached claims. The attached claims and their legal equivalents can be realized in the context of embodiments other than the ones used as illustrative examples in the description below.

(6) The present invention discloses a hydrocarbon refinery wastewater bio-treatment process and more specifically to a process for bio-sludge reduction in effluent treatment plant (ETP) aeration tank through microbial intervention.

(7) In accordance with the present invention a microbial consortia of hydrophilic petroleum hydrocarbon degrading bacterium was prepared. The member used of consortia can be individually-immobilized using artificial inorganic shells, based on methods reported in the literature or as such i.e., free without immobilization.

(8) These microbes as such or after appropriate surface modification can act as an effective bacteria-based emulsifier and/or degrader. When compared to unmodified bacterial cells, these individually-immobilized microbes may act as an effective emulsifier to adsorb at the oil-water interface and then facilitate the biodegradation of oil. This is very desirable for bioremediation of waste water.

(9) The main object of the present invention is to develop a high cell density novel formulation of microbial consortium having following characteristics like, degradation of aromatics, polyaromatics, hetrocyclic polyaromatics, aliphatics, benzene, toluene, ethylbenzene and xylene (BTEX), paraffin, phenolics, sulfides, production of biosurfactants, reduction of toxic metals like hexavalent chromium to non-toxic states, tolerance to hydrocarbon loads. Beside that each microbes should be catalase positive, lipase positive and indole positive.

(10) In accordance with the present invention, each of the microbes in the consortium should contain at least one gene encoding the enzyme/protein/compound responsible for of the above characteristics. Further, each microbe of the consortium should have less expression or should not have expression of the genes responsible for producing exopolysaccharides that cause flocculation.

(11) It is the object of the present invention, that at least one member of microbial consortia disclosed in present invention should have good expression of at least one gene among Lichenan-specific enzyme IIA component (licA), Lichenan-specific enzyme JIB component (licB), Lichenan-specific enzyme IIC component (licC), n-Phosphopantetheinyl transferase (sfp, sfp0), Rhamnosyltransferase subunit A (rh1A), Rhamnosyltransferase subunit B (rh1B), Surfactin synthetase complex (srfAD) for producing biosurfactant/dispersant/surface active molecules. The bacterium included in the present invention should have more than 99% similarity with one of the sequence of SEQ. ID No. 1-4.

(12) The primers and PCR conditions to be used for identifying the gene sequences of SEQ. ID No.1-4 are as follows:

(13) TABLE-US-00001 F5-CGTTCGCTCAGTCATAAGCA-3 R5-CCTGTATGCACACCCATCTG-3 F5GCCCACGACCAGTTCGAC-3 R5CATCCCCCTCCCTATGAC-3 F5-TCCGTTTTTCCTTGTTCACC-3 R5-TCTTTCTGCCACTGCATCAC-3 F5AGGCAAGCAAGCCTCTGGCG-3 R5CTTGTCCGCACAGGCACCGT-3

(14) The PCR conditions to be used to verify the expression of genes of SEQ. ID No.1-4 are as follows: Initial melting at 94 C. for 5 min, then 35 repeated cycles of holding at 94 C. for 25 sec as denaturation, 54 C. for 40 seconds as annealing and extension at 72 C. for 50 seconds, followed by the final elongation by holding the reaction for 6 minutes at 72 C.

(15) It is the object of the present invention, that at least one member of microbial consortia disclosed in present invention should have good expression of at least one gene among Catechol 1, 2 dioxygenase (CatA), Catechol dioxygenase (C12O, C23O), Naphthalene dioxygenase (nahH, nahI) for degrading the poly aromatics/phenolics/heterocyclics. The bacterium included in the present invention should have more than 99% similarity with one of the sequence of SEQ. ID No. 5-6.

(16) The primers and PCR conditions to be used for identifying the gene sequence of SEQ. ID No. 5-6 are as follows:

(17) TABLE-US-00002 F-5TGGCGATGAAGAACTTTTCC-3 R-5AACGTACGCTGAACCGAGTC-3 F-5AAGAGGCATGGGGGCGCACCGGTTCGA-3 R-5TCACCAGCAAACACCTCGTTGCGGTTGCC-3

(18) The PCR conditions to be used to verify the expression of genes of SEQ. ID No.5-6 are as follows: Initial melting at 94 C. for 5 min, then 35 repeated cycles of holding at 94 C. for 25 seconds as denaturation, 54 C. for 40 seconds as annealing and extension at 72 C. for 50 seconds, followed by the final elongation by holding the reaction for 6 minutes at 72 C.

(19) The microbial consortia disclosed in present invention should have good expression of at least one gene among aromatic ring hydroxylating (RHD) genes for breakdown of aromatic rings. The gene expression should match 90-100% with one of the sequence of SEQ. ID No. 7-8.

(20) The primers and PCR Conditions to be used for identifying the gene sequence of SEQ. ID No. 7-8 are as follows:

(21) TABLE-US-00003 F-5CGGCGCCGACAAYTTYGTNGG-3 R-5GGGGAACACGGTGCCRTGDATRAA-3

(22) The PCR conditions to be used to verify the expression of genes of SEQ. ID No.7-8 are as follows: Initial melting at 94 C. for 5 min, then 35 repeated cycles of holding at 94 C. for 25 seconds as denaturation, 54 C. for 40 seconds as annealing and extension at 72 C. for 50 seconds, followed by the final elongation by holding the reaction for 6 minutes at 72 C.

(23) The microbial consortia disclosed in present invention should have good expression of at least one gene among Alkane monooxygenase (AlkB, AlkB1), Alkane dioxygenase (nahAc), Flavin-binding monooxygenase (almA), Long chain alkane monooxygenase (ladA) for degrading aliphatics like n-alkanes, long-chain alkanes, paraffins, etc. The gene expression should match 100% with one of the sequence of SEQ. ID No. 9-11.

(24) The primers and PCR conditions to be used for identifying the gene sequence of SEQ. ID No. 10-11 are as follows:

(25) TABLE-US-00004 F-5AACTACMTCGARCAYTACGG-3 R-5TGAMGATGTGGTYRCTGTTCC3 F-5TACGGGCACTTCGCGATTGA-3 R-5CGCCCAGTTCGAMACGATGTG-3

(26) The PCR conditions to be used to verify the expression of genes of SEQ. ID No.10-11 are as follows: Initial melting at 94 C. for 5 minutes, then 35 repeated cycles of holding at 94 C. for 25 seconds as denaturation, 54 C. for 40 seconds as annealing and extension at 72 C. for 50 seconds, followed by the final elongation by holding the reaction for 6 minutes at 72 C.

(27) The microbial consortia disclosed in present invention should have good expression of at least one gene among quinone oxidoreductase (sqr), Persulfide dioxygenase (pdo), for sulfide removal. The microbial consortia disclosed in present invention should have good expression of Catechol 2, 3 dioxygenase (xy1E) gene responsible for BTEX degradation. The microbial consortia disclosed in present invention should have good expression of at least one gene among Chromate Reductase (ChrR) for chromium reduction.

(28) Further, the microbial consortia disclosed in present invention should not have expression of any gene among Transcriptional activator protein (flcA, LuxR/UhpA) genes for suppressing the exopolysaccharides production/flocculation. The gene expression should match 99% with the sequence of SEQ. ID No. 12-15.

(29) The primers and PCR conditions to be used for identifying the gene sequence of SEQ. ID No. 12-15 are as follows:

(30) TABLE-US-00005 F-5TACGGGCACTTCGCGATTGA-3 R-5CGCCCAGTTCGAMACGATGTG-3

(31) The PCR conditions to be used to verify the expression of genes of SEQ. ID No. 12-15 are as follows: Initial melting at 94 C. for 5minutes, then 35 repeated cycles of holding at 94 C. for 25 seconds as denaturation, 54 C. for 40 seconds as annealing and extension at 72 C. for 50 seconds, followed by the final elongation by holding the reaction for 6 minutes at 72 C.

(32) In an embodiment, a wastewater treatment system includes a reactor comprising a wastewater input, a bacteria input, a nutrients input, an oxygen input. In another embodiment the method of treating wastewater comprises the steps of: i. continuously flowing wastewater into a reactor; ii. continuously dosing microbes in log phase; and iii. continuously discharging clean water out of the reactor.

(33) In an embodiment of the said process includes a wastewater input, bacteria and nutrients input, an oxygen input, and a clean water output. In another embodiment, the said wastewater bio-treatment process runs at a mixed liquor suspended solids (MLSS) concentration of 1000 mg/L. In yet another embodiment the said wastewater bio-treatment process does not require recycling of the biomass to the aeration tank.

(34) In a further embodiment of the present invention, the bio-sludge production is at least 50% less than conventional activated sludge process. In yet another embodiment, the retention time of the bacteria in the aeration tank is 4-16 hours. The microbial consortia used in the present invention dose is tolerant to shock loads and does not requires the recycling of the biomass, hence, upset can be managed effectively.

(35) In an embodiment of the present invention, the ratio of Pseudomonas sp. to Bacillus on a biomass basis, is desirably from about 10:1 to about 1:10, preferably from about 5:1 to about 1:5, and most preferably from about 2:1 to 1:2.

(36) The microbial consortia disclosed in present invention may include at least one Pseudomonas species and one Bacillus species. Species of Pseudomonas are selected from the group consisting of Pseudomonas stutzeri (MTCC 25027), Pseudomonas aeruginosa (MTCC 5389), Pseudomonas aeruginosa strain IOC DHT (MTCC, 5385), Pseudomonas putida IOCR1 (MTCC 5387), Pseudomonas putida IOC5a1 (MTCC 5388) and a mutant of any one of said microbes possessing all the identifying characteristics of any one of said microbes, and mixtures thereof.

(37) Species of Bacillus are selected from the group consisting of Bacillus subtilis (MTCC 25026), Bacillus substilis (MTCC 5386), Bacillus thermoleovorans (MTCC 25023), Bacillus stearothermophilus (MTCC 25030), Lysinibacillus sp. (MTCC 25029), Lysinibacillus sp. (MTCC 5666) and a mutant of any one of said microbes possessing all the identifying characteristics of any one of said microbes, and mixtures thereof.

(38) The microbial consortia disclosed in present invention may include at least one Pseudomonas and one Bacillus where at least one microbe has ability to produce surface active molecule. The microbes disclosed in the present application have constitutive expression of various hydrocarbon degrading genes and at least one microbe has ability to produce dispersant/surface active molecules.

(39) The microbial consortium of the invention has been found to be highly effective for rapidly metabolizing and eliminating a wide variety of hydrocarbons. The mixed culture and methods of the invention have the additional advantage of being totally innocuous to the environment. The bacteria and nutrients have no adverse effect on the ecosystem either during or after the bio-treatment process. The bacteria generally die, or at least their population is reduced to a minuscule level, after the hydrocarbons in the system are consumed. The hydrocarbon contaminants are completely mineralized by the mixed bacteria culture of the invention, under aerobic conditions, yielding harmless products (e.g. carbon dioxide, water, biomass and salts).

(40) In accordance with another aspect of the current invention, all the microbes were selected for stress tolerance. The stress factors included were acidity, alkalinity, high temperature and low temperature. These strains show growth profile under broad range of temperature (5 C. to 40 C.) and pH (4.0 to 9.0).

(41) A suitable nutrient system used in the process is disclosed comprising a nitrogen source, such as an ammonium salt, and a phosphorus source, such as an alkali metal phosphate compound. The nutrient system desirably includes a magnesium source, such as a magnesium salt, and can optionally include other nutrients such as sodium, calcium and iron salts.

(42) For growth of microbes in the tank at inlet of the aeration tank, the foregoing nutrients are dissolved in a suitable amount of water to dissolve the nutrients and combined with appropriate quantities of a suitable initial primary food source and the mixed bacteria culture. A suitable initial primary food source is generally organic sugar and/or any aliphatic or mononuclear aromatic hydrocarbon. The improvement provided by the present invention involves a controlled-release source of microbial nutrients at a low level and optionally vitamins and/or nutrients which double as buffering agents to keep the surrounding environment of the product at a pH which is compatible with the growth and activity of the particular microbes.

(43) In accordance with the present invention, a method for isolating a useful microbial consortium comprises the steps of: Isolating the bacterium. developing of an synergistic mixture

(44) According to the method of the present application the reactor is a continuously stirred reactor. According to the method of present application the reactor is working when DO is at least 3 ppm. According to the method of present application, the method is workable when the microbial count of the introduced microbe is at least 10.sup.2 cful/ml.

(45) The method of present application leads to reduction in: chemical oxygen demand (COD), total petroleum hydrocarbons (TPH), biochemical oxygen demand (BOD), total organic carbon (TOC), total suspended solids (TSS), sulfide, phenol, poly aromatic hydrocarbons (PAH), volatile suspended solids (VSS), total carbon (TC), total inorganic carbon (TIC), oil and grease content of the input wastewater. According to the method of present application, at least one of the first bioreactor and the second bioreactor is at aerobic conditions.

(46) Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiment thereof.

Example-1

(47) Microbial consortium comprising of the aforesaid characteristics was prepared using Pseudomonas stutzferi (MTCC 25027), Bacillus subtilis (MTCC 25026) Bacillus substilis (MTCC 5386), Pseudomonas aeruginosa (MTCC 5389), Lysinibacillus sp. (MTCC 5666), Lysinibacillus sp. (MTCC 25029) are used as example.

(48) The said microbial blend has a very good expression of Surfactin synthetase complex (srfAD) and Phosphopantetheinyl transferase (sfp) genes for producing biosurfactants called, surfactin. The consortium also has good expression of Catechol 1, 2 dioxygenase (catA), Naphthalene dioxygenase (nahH), Alkane monooxygenase (alkB and alkB1) genes for degrading the aromatics and aliphatics respectively. The consortium also has very good expression of Long chain alkane monooxygenase (ladA) for degrading the long chain alkanes/paraffins. The consortium also has a good expression of Persulfide dioxygenase (pdo) gene, Catechol 2, 3 dioxygenase (xylE) and Chromate Reductase (chrR) genes for sulfide removal, BTEX degradation and chromium reduction. The consortium has very weak expression of Transcriptional activator protein (flcA) gene responsible for flocculation.

(49) The microbial consortia were evaluated to decontaminate the refinery wastewater in a continuous CSTR. The performance of the consortia was compared with activated sludge obtained from the refinery. The performance comparison is as follows:

(50) TABLE-US-00006 Outlet Microbial Microbial consortia of consortia Activated present disclosed in WO Parameter Inlet sludge invention 2014/033638 A2 Oil & Grease (ppm) 555 08.7 04.1 5.0 Phenol (ppm) 12 1.23 0.26 0.30 Sulphide (ppm) 23 1.74 0.25 0.30 MLSS (ppm) 3546 980 4500 Bio-sludge generation 7.69 0.98 6.17 in treating 100 L water (g, dry weight)

Example-2

(51) The microbial consortia consisting of Bacillus subtilis (MTCC 25026) Bacillus substilis (MTCC 5386), Pseudomonas aeruginosa (MTCC 5389), Bacillus stearothermophilus (MTCC 25030), Lysinibacillus sp. (MTCC 5666), were evaluated to decontaminate the refinery wastewater in a continuous CSTR. The microbial count was maintained at 10.sup.2 cfu/ml. The ratio of Pseudomonas and Bacillus was maintained 2:10. The performance of the consortia was compared with activated sludge obtained from the refinery. The performance comparison is as follows:

(52) TABLE-US-00007 After treatment Before Activated Microbial consortia Parameter treatment sludge disclosed in this invention O&G (ppm) 555 08.7 3.95 Phenol (ppm) 12 1.23 0.22 Sulphide (ppm) 23 1.74 0.28 MLSS (ppm) 3546 1030 Bio-sludge generation 7.69 1.16 in treating 100 L water (g, dry weight)

Example-:3

(53) The microbial consortia consisting of Pseudomonas stutzferi (MTCC 25027) Bacillus subtilis (MTCC 25026) Lysinibacillus sp. (MTCC 5666), Pseudomonas putida IOCR1 (MTCC 5387), Lysinibacillus sp. (MTCC 25029) were evaluated to decontaminate the refinery wastewater in a continuous CSTR. The microbial count was maintained at 10.sup.4 cfu/ml. The ratio of Pseudomonas and Bacillus was maintained 1:10. The performance of the consortia was compared with activated sludge obtained from the refinery. The performance comparison is as follows:

(54) TABLE-US-00008 After treatment Microbial Activated consortia disclosed Parameter Inlet sludge in this invention O&G (ppm) 555 08.7 1.2 Phenol (ppm) 12 1.23 0.09 Sulphide (ppm) 23 1.74 0.06 MLSS (ppm) 3546 720 Bio-sludge generation 7.69 0.86 in treating 100 L water (g, dry weight)