1. Bioremediation

2. Bioremediation ? Biology + Remediation = Bioremediation Biological organisms (bacteria, fungi, plant) Method used to clean the contaminated area High toxic to less toxic (or) non toxic

3. Principles Microorganisms - take pollutants from the environment - used to enhance the growth and metabolic activity Bacteria, Fungi are well known for degrading complex molecules and transform the product into part of their metabolism

4. Definition The process whereby organic wastes are biologically degraded under controlled conditions to an innocuous state. Bioremediation is the use of living organisms, primarily microorganisms, to degrade the environmental contaminants into less toxic forms.

5. Process Microorganisms release enzymes to breakdown the contaminant into digestible farm

6. BIOREMEDIATION In situ Ex situ Bioventing Biosparging Biostimulation Bioaugmentation Phytoremediation Land farming Compost Biopiles Bioreactors

7. Bioventing The most common in situ treatment Supplying air and nutrients through wells to contaminated soil to stimulate the indigenous bacteria. Bioventing employs low air flow rates and provides only the oxygen necessary for the biodegradation while minimizing volatilization and release of contaminants to the atmosphere. It works for simple hydrocarbons and can be used where the contamination is deep under the surface.

8. Bioventing

9. Biosparging Biosparging involves the injection of air under pressure below the water table to increase groundwater oxygen concentrations and enhance the rate of biological degradation of contaminants by naturally occurring bacteria. Biosparging increases the mixing in the saturated zone and there by increases the contact between soil and groundwater. Low cost of installing small - diameter air injection points allows considerable flexibility in the design and construction of the system.

10. Biosparging

11. Biostimulation It involves supplying oxygen and nutrients by circulating aqueous solutions through contaminated soils to stimulate naturally occurring bacteria to degrade organic contaminants. It can be used for soil and groundwater. Generally, this technique includes conditions such as the infiltration of water - containing nutrients and oxygen.

12. Bioaugumentation Bioremediation frequently involves the addition of microorganisms indigenous or exogenous to the contaminated sites. Two factors limit the use of added microbial cultures in a land treatment unit: 1) nonindigenous cultures rarely compete well enough with an indigenous population to develop and sustain useful population levels and 2) most soils with long-term exposure to biodegradable waste have indigenous microorganisms that are effective degrades if the land treatment unit is well managed.

13. Land forming It is a simple technique in which contaminated soil is excavated and spread over a prepared bed and periodically tilled until pollutants are degraded. The goal is to stimulate indigenous biodegradative microorganisms and facilitate their aerobic degradation of contaminants. In general, the practice is limited to the treatment of superficial 10–35 cm of soil. Since landfarming has the potential to reduce monitoring and maintenance costs, as well as clean-up liabilities, it has received much attention as a disposal alternative.

14. Composting Composting is a technique that involves combining contaminated soil with nonhazardous organic amendants such as manure or agricultural wastes. The presence of these organic materials supports the development of a rich microbial population and elevated temperature characteristic of composting.

15. Biopiles Biopiles are a hybrid of landfarming and composting. Essentially, engineered cells are constructed as aerated composted piles. Typically used for treatment of surface contamination with petroleum hydrocarbons they are a refined version of landfarming that tend to control physical losses of the contaminants by leaching and volatilization. Biopiles provide a favorable environment for indigenous aerobic and anaerobic microorganisms.

17. Bioreactors Slurry reactors or aqueous reactors are used for ex situ treatment of contaminated soil and water pumped up from a contaminated plume. Bioremediation in reactors involves the processing of contaminated solid material (soil, sediment, sludge) or water through an engineered containment system. A slurry bioreactor may be defined as a containment vessel and apparatus used to create a three - phase (solid, liquid, and gas) mixing condition to increase the bioremediation rate of soil bound and water-soluble pollutants as a water slurry of the contaminated soil and biomass (usually indigenous microorganisms) capable of degrading target contaminants.

18. Phytoremediation Plants have been commonly used for the bioremediation process called Phytoremedation, which is to use plants to decontaminated soil and water by extracting heavy metals or contaminants. Plants that are grown in polluted soil are specialized for the process of Phytoremedation. The plants roots can extract the contaminant, heavy metals, by one of the two ways, either break the contaminant down in the soil or to suck the contaminant up, and store it in the stem and leaves of the plant. Usually the plant will be harvest and removed from the site and burned. Phytoremediation process is used to satisfy environmental regulation and costs less then other alternatives. This process is very affective in cleaning polluted soil.

19. Types Phytoextraction Phytotransformation Phytostabilisation Phytodegradation Rhizofiltration

20. Phytoextraction The plants to accumulate contaminants into the roots and aboveground shoots or leaves. This technique saves tremendous remediation cost by accumulating low levels of contaminants from a widespread area. Unlike the degradation mechanisms, this process produces a mass of plants and contaminants (usually metals) that can be transported for disposal or recycling.

21. Phytotransformation Refers to the uptake of organic contaminants from soil, sediments, or water and, subsequently, their transformation to more stable, less toxic, or less mobile form. Metal chromium can be reduced from hexavalent to trivalent chromium, which is a less mobile and noncarcinogenic form.

22. Phytostabilization The plants reduce the mobility and migration of contaminated soil. Leachable constituents are adsorbed and bound into the plant structure. They form a stable mass of plant from which the contaminants will not reenter the environment.

23. Phytodegradatio n Breakdown of contaminants through the activity existing in the rhizosphere. This activity is due to the presence of proteins and enzymes produced by the plants or by soil organisms such as bacteria, yeast and fungi. Rhizodegradation is a symbiotic relationship that has evolved between plants and microbes. Plants provide nutrients necessary for the microbes to thrive, while microbes provide a healthier soil environment.

24. Rhizofiltration It is a water remediation technique that involves the uptake of contaminants by plant roots. Rhizofiltration is used to reduce contamination in natural wetlands and estuary areas.

25. Limitations of Bioremediation Contaminant type & Concentration Environment Soil type condition & Proximity of ground water Nature of organism Cost benefit ratios : Cost Vs Env. Impact Does not apply to all surface Length of bioremediation process

26. Advantages Minimal exposure of on site workers to the contaminant Long term protection of public health The Cheapest of all methods of pollutant removal The process can be done on site with a minimum amount of space and equipment Eliminates the need to transport of hazardous material Uses natural process Transform pollutants instead of simply moving them from one media to another Perform the degradation in an acceptable time frame

27. DISADVANTAGES Cost overrun Failure to meet targets Poor management Climate Issue Release of contaminants to environment Unable to estimate the length of time it’s going to take, it may vary from site.

28. Bacterial genera isolated from water and sediment samples of different lakes 55 isolates

29. Screening of nitrate reducers No reduction : - Less reduction : + Moderate reduction : ++ High reduction : +++ Nitrate reduction test Reduction of nitrate / nitrite to ammonium Appearance of reddish orange colour Based on intensity of the colour

30. Potent isolates used for study (+++) Pseudomonas sp. (KW 1) Pseudomonas sp. (KW 8) Bacillus sp. (KS 1) Alcaligenes sp. (KS 3) Pseudomonas sp. (KS 5) Pseudomonas sp. (KS 7) Corynebacterium sp. (OW 1) Pseudomonas sp. (OW 6) Bacillus sp. (OW 8) Alcaligenes sp. (OS 1) Alcaligenes sp. (OS 6) Pseudomonas sp. (OS 9) Bacillus sp. (YW 1) Bacillus sp. (YW 4) Bacillus sp. (YW 7) Alcaligenes sp. (YS 5) and Bacillus sp. (YS 8). Out of 55 isolates 17 isolates was found to be potent in nitrate reduction

31. Nitrate reducing efficiency of bacteria in synthetic medium with 100 mg.L -1 of nitrate at 48 hrs Growth - 95 x 10 3 cfu.mL -1 (KW1) NO 3 - 80.2%, 78.9% (KW1, YW4) Nitrite - 0.75 mg.L -1 (YS8) Ammonium - 2.8 mg.L-1 (OS 1)

32. Based on the above results (> 70%) the following isolates were selected for further analysis of nitrate removal. A - Pseudomonas sp. (KW 1) B - Bacillus sp. (KS 1) C - Corynebacterium sp. (OW 1) D - Pseudomonas sp. (OW 6) E - Bacillus sp. (OW 8) F - Alcaligenes sp. (OS 1) G - Pseudomonas sp. (OS 9) H - Bacillus sp. (YW 4)

33. Where A. Pseudomonas sp. (KW 1) B. Bacillus sp. (KS 1) C. Corynebacterium sp. (OW 1) D. Pseudomonas sp. (OW 6) E. Bacillus sp. (OW 8) F. Alcaligenes sp. (OS 1) G. Pseudomonas sp. (OS 9) H. Bacillus sp. (YW 4) A + BA + B + CA + B + C + D A + CA + B + DA + B + C + E A + DA + B + EA + B + C + F A + EA + B + FA + B + C + G A + FA + B + GA + B + C + H A + GA + B + HB + C + D + E A + HB + C + DB + C + D + F B + CB + C + EB + C + D + G B + DB + C + FB + C + D + H B + EB + C + GC + D + E + F B + FB + C + HC + D + E + G B + GC + D + EC + D + E + H B + HC + D + FC + D + E + A C + DC + D + GD + E + F + G C + EC + D + HD + E + F + H C + FC + D + AD + E + F + A C + GD + E + FD + E + F + B C + HD + E + GE + F + G + H D + ED + E + HE + F + G + A D + FD + E + AE + F + G + B D + GD + E + BE + F + G + C D + HE + F + GF + G + H + A E + FE + F + HF + G + H + B E + GE + F + AF + G + H + C E + HE + F + BF + G + H + D F + GE + F + CG + H + A + B F + HF + G + HG + H + A + C G + HF + G + AG + H + A + D F + G + BG + H + A + E F + G + CH + A + B + D F + G + DH + A + B + E H + A + B + F Consortium used for the study

34. Nitrate reduction by consortium >86% - A+H 45 % - E+F+C < 29 % - Four

35. From the study A+H (Pseudomonas sp. (KW1) and Bacillus sp. (YW4)) consortia showed maximum nitrate reduction Selected for further kinetic studies (carbon sources, temperature, pH, inoculum dosage) on nitrate removal

36. Effect of various carbon sources on nitrate removal 86 x 10 4 - Starch 100-0.6 mg.L -1 - Starch Starch - Less 99.4 % reduction

37. Effect of various temperatures on nitrate removal in MSM High Temp - Decreese 30 o C High reduction > 99 % - 30 o C

38. Effect of various pH on nitrate reduction by bacterial consortium (A+H) in synthetic medium with 100 mg.L -1 of nitrate 6,9 – less, Max-7 (84.5 x 10 4 ) 6,9 – less, Max-7 (0.6) 6,9 – less, Max-7 (99%) 9 - less, Max-7 6 - less, Max-8

39. Effect of various cell concentrations of bacterial inoculum (A+H) Max- 5% (105), Less – 1% (85) Max- 5% (0.5 mgL -1 ) 5% - More 2 % - max 5% - 99.8, 1%-99.3

40. Drinking water (10 L) + 100 mg.l -1 of NO 3 + Starch (1.0 %) at pH 7 Inoculum (A+H) dosage (1 %) Reactor (18-20 hrs) Settling tank (Coagulants - 15 / 60 min) Sand filter Estimation - Bacterial growth, NO 3, NO 2 and NH 4 6, 12, 18, 24, 30, 36, 42 and 48 hrs Treated water tank

41. Reactor tank Settling tank Filtration tank (55 cm, 60 cm 35 cm) Collection tank Reservoir Pilot scale treatment plant (10 litres)

42. Pilot scale study for nitrate removal in drinking water 85 x 10 4 – Max 100-0.5 3.2 - Nitrite 8.4 - Ammonium 99 % - Nitrate

43. Large scale study in nitrate reduction in drinking water sample (A+H)

44. Drinking water (1000 L) + 100 mg.l -1 of NO 3 + Starch (1.0 %) at pH 7 Inoculum (A+H) dosage (1 %) Reactor (18-20 hrs) Settling tank (Coagulants - 15 / 60 min) Sand filter Estimation - Bacterial growth, NO 3, NO 2 and NH 4 6, 12, 18, 24, 30, 36, 42 and 48 hrs Treated water tank

45. Large scale treatment plant setup used for the study ( IVC labs & Environmental Services, Chennai) 1000 L 10L Package of filter tank Large pebbles : 2.0 - 2.5 cm (bottom) Small pebbles : 0.7 - 1.5 cm Gravel : 0.4 - 0.6 cm Coarse sand : 0.05 - 0.1 cm Fine sand : 0.15 -0.3 mm Activated carbon : 0.75 - 1.0  m Aeration 75 rpm for 16 – 20 hrs

46. Large scale treatment plant (1000 litres)

47. Every six hours upto 48 hrs water sample was analysed for NH 4, NO 2 and NO 3. The water was collected from the collection tank after treatment and was subjected to its physico-chemical and bacteriological quality and compared with the standards for drinking water.

48. Large scale study for nitrate removal by bacterial consortium (A + H) in drinking water 74 x 10 4 – Max 100-8 2.1 – Nitrite 7.6- Ammonium 92%

49. Physico-chemical parameters of water sample before and after large scale treatment Parameters Untreated water Treated water ISI drinking water standard pH7.17.36.5 - 8.5 Conductivity ( mS) 1110- Turbidity (NTU)725 OdourNone Unobjectionable Total solids855240500 Total hardness10815300 Chloride136250 Nitrate100845 Sulphate0.40.05200 Phosphate120.4- THB (CFU.mL -1) 74 x 10 4 16 x 10 1 - All the values are expressed in mg.L -1 except pH, EC and turbidity.

51. Lab scale anaerobic reactor

53. 100 ℓ Lab scale 30 ℓ Lab scale Feeding Mode Intermittent Feeding Continuous feeding RatioFW FW+ SS (1:9) FW+ SS (1:9) FW+ SS (2:8) FW+ SS (3:7) FW+ SS (4:6) FW+ SS (5:5) SS only (0:10) Days 1 – 2930 – 8687 – 118119 – 139140 – 172173 – 203204 – 264265- 356 Digester 1 (100 L) HRT20 days15 days10 days5 days Days 1 – 182183 – 244245 –300301-336 Shock load 20 o C (84 th day) 40 o C (99 th day) 45 o C (155 th day) Digester 2 (Temperature shock test - days)

54. Parameters FW+ SS (1:9) FW+ SS (2:8) FW+ SS (3:7) FW+ SS (4:6) FW+SS (5:5) FW+SS (0:10) pH 7.47 (7.19 - 7.61) 7.58 (7.52 – 7.64) 7.70 (7.56 – 7.85) 7.71 (7.64 – 7.81) 7.88 (7.84 – 7.92) 7.63 (7.59 – 7.65) Biogas (L/Day) 43.8 (37 - 46) 47.23 (46 - 48) 47.76 (46 – 48) 60.37 (58 - 64) 85.18 (84 - 86) 33.24 (32 - 34) CH 4 (L/Day) 31.49 (27 - 38) 34.73 (33 - 35) 33.32 (31 - 35) 40.07 (34 - 46) 60.07 (57 – 63) 23.72 (21 - 24) CH 4 (L/g VS) 0.162 (0.148–0.169) 0.214 (0.204–0.241) 0.210 (0.192– 0.241) 0.240 (0.193– 0.282) 0.268 (0.251–0.304) 0.195 (0.175–0.210) CH 4 (L/g TCOD) 0.284 (0.213-0.328) 0.381 (0.343-.0411) 0.401 (0.377-0.470) 0.240 (0.179-0.304) 0.371 (0.340-.0389) 0.234 (0.208-0.272) VS red (%) 49.97 (44.4 – 61.5) 43.59 (39.1 – 52.5) 53.45 (48.8 – 58.7) 57.79 (53.7 – 65.2) 64.98 (64.2 – 68.5) 35.38 (32.2 – 41.2) SCOD red (%) 60.71 (50.0 – 66.6) 61.44 (51.2 – 69.4) 60.04 (53.1 – 66.6) 68.12 (58.8 – 74.4) 63.57 (56.5 – 66.2) 46.72 (41.1 – 47.3) TCOD red (%) 58.34 (48.2 – 66.6) 67.69 (53.9 – 75.5) 62.97 (57.4 – 68.6) 53.04 (47.3 – 56.6) 61.58 (57.4 – 67.1) 56.9 (53.8 – 60.0)

55. ParametersFW+ SS(1:9)FW+ SS (2:8)FW+ SS (3:7)FW+ SS(4:6)FW+SS(5:5) FW+SS (0:10) SCOD(mg/L) Inf 14544 (11040-19680) 12480 (11200-16640) 11440 (10240-12480) 13668 (11200-16640) 15197 (14080-15936) 12164 (11832-12268) SCOD(mg/L) 5163 (3840-5760) 4891 (3840-6720) 4680 (4160-5440) 4342 (3200-6080) 5591 (5248-6720) 6544 (5452-6912) TCOD(mg/L) Inf 17978 (14400-22080) 18880 (15360-20480) 17208 (15360-20480) 30308 (24320-33920) 32307 (30720-33920) 20753 (18345-21625) TCOD(mg/L) 7040 (5280-8320) 6293 (3520-8320) 6080 (5440-7360) 14125 (12800-15680) 12370 (10800-13944) 8868 (8352-9766) Alkali(mg/L) Inf 708 (642 - 940) 535 (480 - 620) 424 (380 - 480) 352 (260 - 428) 320 (260 - 460) 1592 (1480 - 1680) Alkali(mg/L) 3041 (2740 - 3880) 3066 (2920 - 3160) 3250 (2960 - 3249) 3786 (3660-3880) 5326 (4240 - 5868) 4557 (4500 - 4700) VFA(mg/L) Inf 660 (586 - 743) 813 (689 - 843) 830 (798 - 956) 3415 (3400 – 3865) 4731 (4234 - 5122) 3060 (3024 - 3124) VFA(mg/L) 259 (214 - 350) 319 (297 - 371) 357 (267 - 423) 395 (278 - 475) 503 (456 - 521) 344 (326 - 365)

59. ParametersHRT 20 daysHRT 15 daysHRT 10 daysHRT 5 days pH 7.84 (7.81-7.87) 7.76 (7.75-7.79)7.85 (7.82-7.89) Biogas(L/Day) 12.92 (12.5-13.3)13.40 (13.1-13.8)15.46 (15.1-15.9)17.24 (16.8-17.8) Methane(L/Day) 8.77 (7.37-9.80)9.36 (9.03-10.05)10.87 (10.85-11.28)12.48 (11.45-13.97) CH4(L/g VS) 0.191 (0.138-0.236)0.231 (0.196-0.265)0.181 (0.170-0.177)0.120 (0.111-0.130) CH4 (L/g TCOD) 0.191 (0.146-0.235)0.204 (0.188-0.221)0.159 (0.147-0.177)0.108 (0.102-0.119) VS red (%) 45.81 (43.47-48.3)61.44 (58.4-63.2)61.46 (57.3-66.2)54.2 (55.2-56.9) SCOD red (%) 66.32 (62.5-76.08)61.34 (59.09-61.98)57.77 (51.72-63.33)60.09 (58.33-63.88) TCOD red (%) 52.30 (43.19-56.52)62.06 (60.0-63.82)58.83 (54.54-64.58)56.04 (52.83-56.60) SCOD (mg/L) inf 15104 (12800-16000)13937 (13440-14432)20385 (19532-21377)25306 (25113-26107) SCOD mg/L 5056 (3580-5760)5360 (4816-5904)8596 (7673-9676)10092 (9193-10905) TCOD(mg/L) inf 28456(27840-31150)30710 (29520-32336)33692 (32089-37324)37342 (36633-37488) TCOD mg/L 13632 (12800-17600)11733 (11424-11952)13868 (11723-16588)16408 (15312-17680) Alkali (mg/L) inf 659.5 (578-720)1449.2 (1420-1460)1620 (1580-1640)1615 (1596-1626) Alkali (mg/L) 4190 (3900-4640)4523 (4308-4680)4472 (4260-4680)5189 (5004-5290) VFA (mg/L) inf 595.6 (567-638)577.5 (548-604)675.04 (645.67-713)779.5 (768.3-798.6) VFA - mg/L 305.5 (297-324)328.27 (312.4-346.1)354.40 (346.5-375)360.97 (345.5-386.7) HRT Effect from 30L digester

66. Saturday, December 22, 2007 Bioremediation patent for Prof. K.M. Elizabeth Prof. K.M. Elizabeth of the Department of Microbiology in Gitam University has succeeded in ammonia removal from industrial effluents through a bio-remediation method and has obtained a patent for the method, which will be of use in steel industry in particular. The inventor claims ' the bacterium identified by him can remove 100 per cent ammonia within 24 hours, according to Nessler’s method, and 75 per cent according to the Russian method of Nesselerisation'. This is an indication of quality research work done in lesser known universities.Gitam University patentclaims

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