The process of bioremediation has many stages involved in it:
- The selection of microorganism that have the specific ability to detoxify unfavorable chemicals.
- Development of enzymes and proteins that can catalytically convert these wastes.
- Bioprocess technology to harness these abilities.
This extended essay presents the case for the utilization of natural biodiversity, of our planet’s greatest but least developed resource for biotechnological innovation. Biotechnology in recent times has been dominated by spectacular developments in recombinant DNA technology, and without question, there has provided unique opportunities for commercialization. Perhaps bioremediation has an untapped commercial value and most definitely is of significant ecological value.
The studies were carried out at Department of Microbiology of Jai Hind College, Mumbai under the guidance of Dr. M. T. Pandya, Professor and Head of The Department. The studies were carried out to demonstrate the process of Bioremediation of Soil and Water samples contaminated with paraffin oil and phenols, using bacteria isolated from unmodified and fresh soil samples.
MATERIALS AND METHODS.
Bacterial species of degrading Paraffin oils (straight Chain hydrocarbon) and Phenols(Aromatic compound) were isolated from Soil by mineral salt enrichment culture cultivation technique using Mineral salt medium.
Enrichment studies were carried out in modified “Bushnell & Haas Medium (1941)” using liquid enrichment technique reported by Doctor Pandya (year). The table below shows the composition.
The pH of the culture medium was 7 - 7.5.
Phenols at 1000ppm and Paraffin at 1% levels were added separately in flasks containing 100ml medium”.
The enriched samples in flasks were incubated at ambient temperature on rotary shaker with speed 250r.p.m.untill visible growth of bacteria was observed in 3-7 days period.
The degradation of paraffin was indicated by a visible growth at the junction of two liquids.
Photograph-I and II shows the flasks of enrichment experiment
The degradation of phenol was tested by using colorimetric method using 4-Aminoantipyrine and Potassium Ferricynide under alkaline condition. (reference-Standard methods for water and wastewater analysis APHA- American public health association publications).
The concentration of phenol in the sample was estimated by taking one ml of sample, 4 ml of borate buffer, 1 ml of 4-Aminoantipyrine and 1 ml of Potassium Ferricynide. Presence of phenol was indicated by a pink colour. The concentration of phenol was determined using a standard graph, plotted using different concentration of phenol (0, 10, 20, 30, 40 and 50 ppm) and the optical density at each concentration respectively at 540 nm.
In an attempt to study the morphology of the bacteria slides were prepared by making a smear of the culture on a blank slide. Care was taken to keep the smear very thin. This slide was then sent to the Jaslok hospital and research center, where the slides were stained using Gram’s staining technique and the stained slides were observed and photographed under a high-resolution microscope with camera.
Photograph-III and IV shows the Morphology of enriched bacteria.
The enriched organisms were isolated on sterile Nutrient agar plates. The plates were incubated at room temperature for 48 hours, till well-isolated colonies were observed.
Photograph-V shows a growth of one of the isolated bacteria on slant of Nutrient Agar.
TABLE 1. NUMBER OF BACTERIAL ISOLATES
The bacterial isolates were then screened for their ability to degrade paraffin and phenols so as to select the best one.
Although the isolated bacteria could grow in media containing both paraffin and phenol but it was observed that the growth was excellent in a mixed form. Therefore a mixed culture containing all the bacteria was used for studying bioremediation in the laboratory
Rate of degradation of phenol was tested using COD (chemical oxygen demand) and colorimetric methods.
Chemical Oxygen Demand of water sample containing phenols was determined using a “Dichromate reflux method” using “Digestion apparatus with reflux arrangements” as reported in Standard Methods for water and wastewater analysis (APHA publication).
Photograph-VI shows the assembly.
The COD estimations were carried out as foll0ws.
1ml sample diluted to 10ml using distilled water was taken in a clean round bottom 300ml distillation flask.
5ml of Conc.Sulphuric acid was added to the sample along with glass beads.
10ml 0.25N Potassium Dichromate solution was added to the flask.
25ml of Sluphuric acid Silver Sulphate catalyst mixture was then slowly added to the flask.
The flask was connected with a reflux condenser and then transferred to heating mental.
The contents were refluxed for two hrs.
After two hours the contents were thoroughly cooled and diluted with water.
Residual dichromate was titrated using ferroin indicator against 0.1N Ammonium ferrous sulphate solution to a wine red colour end point.
COD values were calculated using flowing formula.
(B-S) xN of Ferrous Ammonium Sulphate x 8000
COD in PPM= ml of Sample.
B- Blank reading. S- Sample Reading
The degradation of paraffin was judged by using growth at the junction of two liquids.
Degradation of Phenol in Water.
In a 1 liter working capacity reactor with aeration arrangement 900ml of water was added.
Phenol solution was added so as to get a concentration of 1000ppm.
Water with phenol was supplemented with urea nitrogen, buffer, calcium, Magnesium and Ferric ions. pH was maintained in neutral range.
100ml of the developed bacterial culture was added to the reactor.
Reactor was then aerated continuously.
Samples were collected at 24hrs interval until 96hrs.
Samples were analysed for COD and residual phenols by methods as mentioned above.
Table-2 shows the results.
The studies were quite interesting and I wanted to carry out more work, however the experimental work requires lot of expertise and at my level it is difficult for me to do the same.
During the course of the experimental work I visited a small lake near Borivali (a district of Mumbai, India) temple. The level of pollution can be seen in pictures (put numbering)
Degradation of Phenol in Soil.
The degradation of Phenol in soil was studied in a small tray containing 4-5 kg of Garden soil. Phenol was added at concentration of about 1000ppm.
Soil was supplemented with nitrogen and buffer.
Bacteria culture which were grown for 48hrs were added to soil (100ml).
The contents were mixed thoroughly and then samples were analyzed after 24hrs interval.
Samples were tested for residual phenols concentration by using method as described above.
Table-3 shows the residual levels of phenol in soil.
RESULTS AND DISCUSSION
Table-2: Degradation of Phenol in water.
From the above graph it is clear that the bacteria bioremdiate phenol surprisingly fast and in 96 hours (4 days) the phenol is completely degraded. The decreasing chemical oxygen demand also allows one to draw the same conclusion. Thus bioremediation can be carried out at a contaminated site and the contaminant should theoretically degrade completely. But it is important to remember that laboratory conditions are significantly different
Table-3: Degradation of Phenol in Soil.
Bioremediation provides a good cleanup strategy for different types of pollution. Bioremediation provides a technique for cleaning up pollution by enhancing the biodegradation processes that occur in nature. Depending on the site and its contaminants, bioremediation may be safer and less expensive than alternative solutions such as incineration or landfilling of the contaminated materials. It also has the advantage of treating the contamination in place so that large quantities of soil, sediment or water do not have to be dug up or pumped out of the ground for treatment.
Bioremediation Processes
Mechanisms of bioremediation include bioaugmentation in which microbes and nutrients are added to the contaminated site or biostimulation in which nutrients and enzymes are added to supplement the intrinsic microbes. In the injection method, bacteria and nutrients are injected directly into the contaminated aquifer, or nutrients and enzymes, often referred to as 'fertilizer', that stimulate the activity of the bacteria are added. In soil remediation, usually nutrients and enzymes are added to stimulate the natural soil bacteria, though sometimes both nutrients and bacteria are added. When the treatment is stopped, the bacteria die. This technique works best on petroleum contamination.
Versatile metabolic activities of microorganisms has played a key role in biodegradation of various toxic compounds, which find their way into natural environment through industrial waste discharges, accidental spillages and excessive usages.
Wide range of microorganism are capable of degrading organic compounds of both aromatic and aliphatic nature under favorable environmental conditions. Species of Pseudomonas, Micrococcus, Nocardia, Bacillus Corynebacterium, Rhodococcus, ,Arthrobacter,Corynebacterium, Flavobacterium, Cladosporium,Fusarium, Chaectomium,and many more are reported. These microbes are widely distributed in soil, water, sewage, sludge and places where organic compounds are spilled over a long periods.
Microbial removal of degradable organic compounds involve a series of steps including mass transfer, adsorption and biochemical enzymatic reactions leading to growth. During the breakdown the bacteria converts a compound like paraffin to alcohol to aldehyde and to fattyacid which is easily degraded by most of the microorganisms present in natural environment.
In case of phenols first the molecule is converted to dihydroxy derivative leading to the cleavage of the ring.
Several different types of microorganisms were isolated from soil samples, using enrichment culture cultivation technique. Majority of these were bacteria.Table-1 shows the number of potent bacterial isolates with very high activity and growth. The degradation of paraffin were indicated by heavy growth ,emulsification of paraffin and disappearance of red colour in case phenols when tested by using amino antipyrine method.
The growth of enriched bacteria can be seen in the photographs.
Most of these isolates were identified as species of bacteria with cocobacillary to bacillary forms. Both gram positive and gram negative bacteria were present in the enriched mixture as it can be seen from the photograph.
One of the isolated bacterial isolate showed pink coloured growth as it can be seen from the photograph.
Some of these bacteria could be species of Pseudomonas, Micrococcus,and Bacillus.
The efficiency of developed mix bacteria culture was excellent when tested at lab scale level in 1 lit capacity reactor as well as in soil. The trials carried out for phenols showed that in a period of about 96 hrs 1000ppm of phenols was completely removed. COD reductions too were >98% in 96 hrs. In soil samples contaminated with phenols the results were excellent. The bacteria cultures removed Phenols in 96hrs of incubation period.
During the course of experimental work I visited one lake which is contaminated with very poor visibility. This lake can be cleaned by using bacterial culture and proper aeration. Once the water in lake is subjected to remediation programme it will clear and visibility in water will improve to increased depth.
CONCLUSIONS.
In conclusion, in situ bioremediation is the application of biological treatment to the clean up of contaminants in soil, groundwater, and surface waters. During the process, microorganisms, usually bacteria and fungi, feed on the contaminants.They derive nutrition and energy for growth and reproduction. The wastes are used up or converted into a less harmful form, such as water and carbon dioxide.
- Microorganisms created by microbiologists have the potential of helping cleanse the environment of men made pollutants.
- The usefulness of such organisms in pollution abatement depends on compatibility with their environment.
- The experimental work carried out and the results as observed showed the potential of Bacteria in clean up process.
- The biodegradation of pollutants should be using Naturally occurring microorganisms. Hence one should utilize natural organisms to meet growing demands for cleaning operation in the context of an increasing environmental alert society.
- The starting point for biotechnological developments is the search for and discovery of exploitable biological phenomena ; the effective planning and optimization of search & discovery programs is just as crucial as any other phase of biological operation.
- We rely on microorganisms to biodegrade our waste materials. we have came to assume that anything thrown out in the environment will disappear to an incredibly large extent it is true. Microorganisms have a vast capacity for rapid degrading organic materials & thus can be relied upon to act as biological incinerators.
