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ABSTRACT

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ABSTRACT In this present work, the effectiveness of pseudomonas and bacillus on degradation of different oils, especially diesel, petrol, engine oils, etc., were studied. Biological method has been found to be the harmless method for degrading hydrocarbons with low cost and high efficiency. Among the biological methods, bioreactor has been the sophisticated method that can degrade the hydrocarbons to concentrations below the standard limits. Membrane bioreactors can be broadly defined as systems integrating biological degradation of waste products with membrane filtration, with good control of biological activity. The effluent from these bioreactors will be free from chemicals and micro organisms. For the degradation studies, I developed a media using Plackett-Burman design. It has been found to be the easiest and time saving procedure for the development of different media with varying concentrations of nutrients. It offers reduction in BOD within the range of 60-75% and COD reduction up to 83% within 3 days when introduced into the bioreactor. The temperature was maintained at 32oC at a pH of 7.

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INTRODUCTION

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INTRODUCTION The worldwide rise in population and the industrialization during the last few decades have resulted in ecological unbalance and degradation of the natural resources. One of the most essential natural resources which have been the worst victim of population explosion and growing industrialization is water. Today, we withdraw water far faster than it can be rechargedunsustainably mining what was once a renewable resource (Abramovitz, 1996). Huge quantity of wastewater generated from human settlement and industrial sectors accompany the disposal system either as municipal wastewater or as industrial wastewater. This wastewater enriched with varied pollutants and harmful both for human being and the aquatic flora and fauna, finds its way out into the nearly flowing or stationary water bodies and thus make the natural sources of water seriously contaminated. The presence of some harmful pollutants in wastewater deteriorates the water quality considerably and has damaging effect on both aquatic life and human health (B.C. Meikap, Roy, 1995). The worlds rapid population growth over the last century has been a major factor in increasing global water usage. But demand for water is also rising because of urbanization economic development, and improved living standards. Between 1900 and 1995, for example, global water withdrawals increased by over six timesmore than double the rate of population growth ( Gleick ,1998). In developing countries, water withdrawals are rising more rapidlyby four percent to eight percent a year for the past decadealso because of rapid population growth and increasing demand per capita (Marcoux, 1994). Caught between (a) finite and increasingly polluted water supplies, and (b) rapidly rising demand from population growth and development, many developing countries face difficult and uneasy choices. As the World Bank has warned, lack of water is likely to be the major factor limiting economic development in the decades to come ( Serageldin , 1995). In recent years, considerable attention has been paid to industrial wastes discharged to land and surface water. Industrial effluents often contain various toxic metals, harmful dissolved gases, and several organic and inorganic compounds. Huge quantity of waste water generated from human settlement and industrial sectors accompany the disposal system either as municipal wastewater or industrial wastewater (H.M. Jena, et al, 2005). This wastewater is enriched with varied pollutants and harmful both to human being and the

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aquatic flora and fauna and its successive accumulation in the soil has adverse effect on soil productivity. All of Indias 14 major rivers are badly polluted. Together they transport 50 million cubic meters of untreated sewage into Indias coastal waters every year. Indias capital, New Delhi, dumps 200 million liters of raw sewage and 20 million liters of industrial wastes into the Yamuna River every day as it passes through the city on its way to the Ganges ( Harrison,1992).. Environmental contamination due to spills and leaks of petroleum

hydrocarbons from storage facilities and distribution systems has resulted in the contamination of soil and water environments worldwide. Because of the threat they represent to public health, environmental regulations and the need for the safe use of renewable and non-renewable resources, multiple clean up strategies for contamination due to petroleum products have been developed .( Rodrguez-Martnez , 2006). The exploration, production, refining and distribution of petroleum and petrochemical products results in the generation of a considerable volume of waste oil sludges. These sludges come from a variety of sources including storage tank bottoms, oilwater separators, dissolved air floatation units, cleaning of processing equipment, biological sludges from waste water treatment units and oil spills in the oil fields, drilling sites and refineries (Ajay Singh, et al, 2001). The oily sludges are basically composed of oil, water, solids and their characteristics, such as varied composition, make their reutilization very difficult, and confer on them high recalcitrance (Ururahy, et al,1999). A variety of physical, chemical and biological approaches have been taken to remediating refinery sludges. In many countries these sludges have been accumulated in large lagoons, facilitating some recycling of oil but requiring later remediation of residual oily sludges. Attempts to process these sludges using centrifugal methods to separate oil, water and solids phases is highly capital intensive, is not consistently effective and still produces residual solids with high petroleum hydrocarbon content. Another option is to direct oily sludge waste to a delayed coker, however, this can degrade the sludge quality and reduce its economic value. Foul odors are often reported in the coke product after sludge injection and can result in operator complaints. Sludge injection requires modifications to the coker as well as pretreatment of the sludge.

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This pretreatment step requires the use of milling, filtering and centrifugation equipment and a skilled operator. Thermal desorbers and incinerators have been used for the treatment of oily sludges. However, most of the above methods are capital intensive and therefore are associated with overall high sludge treatment costs.(Ajay Singh ,et al,2001). Microbial degradation of hydrocarbons, through either naturally occurring processes or engineered systems, has been successfully used to reduce concentrations of these pollutants to safer environmental levels. Pump and treat systems are one engineering approach that allows the design of a treatment units for optimum biological operation. When combined with fixed film microbial growth, such strategies have shown effectiveness in the processing of sewage and contaminated groundwater. (Enid M. Rodrguez-Martnez, 2006). Oil effluents from different industries such as refineries, petroleum treatment plants and different large scale and small scale industries that are dealing with the petroleum oils contain large amounts of hydrocarbons mainly benzene, toluene, ethyl benzene, xylene. Most of them are highly water soluble and are toxic. When released into water sources, they could cause serious consequences in aquatic flora and fauna and can both directly and indirectly affect humans. Due to the high energy costs, the potential risk of air pollution and the persistence of Polycyclic Aromatic Hydrocarbons (PAHs), incineration is not recommended. Biotreatment can be applied, using the following methods: Composting, Landfarming and Biopile. All of them exploit the soil biodiversity; however they have the disadvantage of needing long process times and there is the risk of contaminating air and aquifiers by leaching. They also demand large areas and are affected by climate. An interesting alternative to this problem is the use of a bioreactor, since optimum process conditions can be easily controlled, allowing higher quality final effluent in shorter times (Ururahy,et al,1999). Several aerobic and anaerobic bioreactors are available for the effective treatment of hydrocarbons from the petroleum effluents. During the last few years, several bacterial cultures have been isolated with the ability to degrade the hydrocarbons (Enid M. Rodrguez-Martnez, 2006). These bacterial cultures when introduced into suitable bioreactor with optimum conditions, within a short period degrade the harmful effluents into harmless

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products, with concentrations well below the recommended limits (Ururahy,et al,1999). The treated water from the bioreactor can be either reused or discharged into the water bodies. MEMBRANE BIOREACTORS Membrane Bioreactors (MBRs) can be broadly defined as systems integrating biological degradation of waste products with membrane filtration. They have proven quite effective in removing organic and inorganic contaminants as well as biological entities from wastewater. Advantages of the MBR include good control of biological activity, high quality effluent free of bacteria and pathogens, smaller plant size, and higher organic loading rates (Cicek,2003). Current applications include water recycling in buildings, municipal wastewater treatment for small communities, industrial wastewater treatment, and landfill leachate treatment. The membrane bioreactor (MBR) process, consisting of an activated sludge bioreactor and a microfiltration membrane, is an emerging and promising technology utilizing a biological treatment process. It takes advantage of the rapid development in membrane manufacturing and has the potential to fundamentally advance the biological treatment of wastewater. The MBR system has exhibited an excellent effluent quality, a high biomass concentration without concern for sludge settling problems, a simple flow configuration, and a small footprint demand. The MBR has been used successfully for biological treatment of wastewater and for the reclamation of treated effluents (Min Jin, et al, 2005). ANAEROBIC BIOREACTORS: As the name indicates, these bioreactors are designed to carry out

biodegradation in the absence of oxygen. The anaerobic process comprises a series of interdependent phases. Initially complex organic compounds such as lipids, proteins and carbohydrates, if present, are hydrolyzed to simpler organics. The latter are then fermented to volatile fatty acids (VFAs) by acidogens .The most common of these fatty acids is ethanoic acid. However, propanoic, butanoic and pentanoic acids may also be present in varying quantities depending on the stability of the process. The acidogens include both facultative and obligate anaerobic bacteria. Subsequent to the acidogenic phase is the methanogenic

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phase. The methanogens are obligate anaerobes and they convert the fatty acids from acidogenesis to methane and carbon dioxide. This results in substantial decrease in the organic content of the wastewater. The methane generated offers an avenue for energy recovery (Buyukgungor, Gurel, 2009). AEROBIC BIOREACTORS: The aerobic biodegradation process is represented by the following equation CxHy + O2 + (microorganisms / nutrient ) ---------- H2O + CO2 + biomass. Aerobic treatment of waste is the degradation and purification process in which bacteria that thrive in oxygen rich environments break down and digest the waste. The mixed aerobic microbial consortium uses the organic carbon present in the effluent as their carbon and energy source. The complex organics finally get converted to microbial biomass (sludge) and carbon di oxide (Behera,2009).

PLACKETT BURMAN DESIGN:

The efficiency of degradation can be increased several fold by using a media that supports the growth of the microorganism. The microorganisms belonging to Pseudomonas species have the ability to use Oil as the sole source of Carbon and energy, which results in degradation of the oil. When it is supplied with a medium that catalyzes its growth, the efficiency can be increased by several folds (Allia, et al, 2006). The cell growth and accumulation of metabolic products in bacteria are strongly influenced by media composition such as carbon sources, nitrogen sources and inorganic salts. Thus it is difficult to search for major factors and to optimize them for biotechnological factors as several factors are involved. The classical method of optimization involves changing one variable at a time by keeping other factors at fixed levels. Statistical method for optimization of media effectively tackles the problem, which involves specific design of experiments which minimizes the error in determining the effect of variables. Placket-Burman design allows reliable short listing of medium components in fermentation for further optimization and allows one to obtain unbiased estimates of linear effects of all factors with maximum accuracy for given number of observations. (Aravindan Rajendran,et al,2007). DEGRADATION OF HYDROCARBONS BY THE MICROORGANISMS

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Biodegradation of petroleum hydrocarbons is a complex process that depends on the nature and on the amount of the hydrocarbons present. Petroleum hydrocarbons can be divided into four classes: the saturates, the aromatics, the asphaltenes (phenols, fatty acids,ketones, esters, and porphyrins), and the resins (pyridines, quinolines, carbazoles, sulfoxides and amides).Several factors influence the hydrocarbon degradation. One of the important factors is their limited availability to the microorganisms.Hydrocarbons differ in their susceptibility to microbial attack. On the basis of susceptibility to degradation, the hydrocarbons can be ranked as follows: linear alkanes > branched alkanes > small aromatics > cyclic alkanes. High molecular weight compounds such as Poly Aromatic Hydrocarbons (PAHs) are not degraded at all. Bacteria are the microorganisms that are highly efficient in degrading petroleum hydrocarbons. Bacteria such as Pseudomonas have the ability to use oil constituents for energy. FACTORS AFFECTING PETROLEUM HYDROCARBON DEGRADATION Several factors limit the degradation of hydrocarbons. One among the important limiting factor is temperature. Decrease in temperature results in lowering the efficiency of biodegradation. Nutrients are very important ingredients for successful biodegradation of hydrocarbon pollutants especially nitrogen, phosphorus and in some cases iron.Another important factor is availability of oxygen. In case of oil spills, the most important organism capable of degradation is Pseudomonas. Among Pseudomonas sp., Pseudomonas putida is the master in degrading oil spills. It has been found that under aerobic conditions, the efficiency of this organism in degradation is increased several folds. Hence aerobic treatment is normally preferred for catalyzing the efficiency of this organism.

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REVIEW OF LITERATURE

BIO-REMEDIATION

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Bioremediation is a powerful technical and scientific approach to alternatively deal with contaminated sites. This process involves the use of microorganisms to degrade organic pollutants such as hydrocarbons, to concentrations that are undetectable or below the limits established as safe to all the living organisms and the environment. (Enid M. Rodrguez-Martnez,2006). BIO TRANSFORMATION Biotransformations involve the use of biological agents, in the form of whole cells or isolated enzymes, to catalyze chemical reactions. Such biotransformation systems may be used for environmentally benign biocatalysis of synthetic reactions, bioremediation of pollutants, or waste beneficiation, a combination of these in which the biological agents convert industrial residues to useful chemical products. In each case, suitable biocatalysts, and suitable bioreactor systems, each with particular characteristics, are required.(Stephanie G. Burton,2001) BIOREACTORS AS USEFUL TOOLS FOR EFFECTIVE ENVIRONMENTAL REMEDIATION As waste management practices become more specific, for particular type of chemical waste, specific treatment system will have to be developed and applied to abate pollution. In near future, since the regulation concerning discharged wastewater will be imposed more strictly, an economical, compact and highly efficient wastewater treatment will be required ( Vijayagopal, Sabarathinam, 2006). Bioreactors have been commonly developed and implemented for bioremediation processes. The goal of bioreactor treatment strategies is to optimize degradation by microbial communities in biofilm or suspended systems in artificially constructed units that allow tightly controlled growth conditions. In suspended growth systems, such as activated sludge, or sequencing batch reactors, the contaminated water is circulated in an aeration basin where microbial populations aerobically degrade the organic matter while CO2, H2O and new cells are produced as degradation products. The cells form sludge, which are settled out in a clarifier unit, and are then either recycled to the aeration basin or disposed of. (Enid M. Rodrguez-Martnez,2006).

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Several aerobic and anaerobic bioreactors are available for the effective treatment of hydrocarbons from the petroleum effluents. During the last few years, several bacterial cultures have been isolated with the ability to degrade the hydrocarbons (Enid M. Rodrguez-Martnez, 2006). These bacterial cultures when introduced into suitable bioreactor with optimum conditions, within a short period degrade the harmful effluents into harmless products, with concentrations well below the recommended limits (Ururahy,et al,1999). The treated water from the bioreactor can be either reused or discharged into the waterbodies

MEMBRANE BIOREACTORS Membrane Bioreactors (MBRs) can be broadly defined as systems integrating biological degradation of waste products with membrane filtration. They have proven quite effective in removing organic and inorganic contaminants as well as biological entities from wastewater. Advantages of the MBR include good control of biological activity, high quality effluent free of bacteria and pathogens, smaller plant size, and higher organic loading rates (Cicek,2003). Current applications include water recycling in buildings, municipal wastewater treatment for small communities, industrial wastewater treatment, and landfill leachate treatment. The membrane bioreactor (MBR) process, consisting of an activated sludge bioreactor and a microfiltration membrane, is an emerging and promising technology utilizing a biological treatment process. It takes advantage of the rapid development in membrane manufacturing and has the potential to fundamentally advance the biological treatment of wastewater. The MBR system has exhibited an excellent effluent quality, a high biomass concentration without concern for sludge settling problems, a simple flow configuration, and a small footprint demand. The MBR has been used successfully for biological treatment of wastewater and for the reclamation of treated effluents (Min Jin, et al, 2005).

COMPONENTS OF A MEMBRANE BIOREACTOR:

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Membrane bioreactors are composed of two primary parts: 1) The biological unit responsible for the biodegradation of the waste compounds, and 2) The membrane module for the physical separation of the treated water from mixed liquor. TYPES OF MEMBRANE BIOREACTORS: MBR systems can be classified into two major groups according to their configuration. 1) The first group, which is also commonly known as the integrated MBR, involves outer skin membranes that are internal to the bioreactor. The driving force across the membrane is achieved by pressurizing the bioreactor or creating negative pressure on the permeate side of the membrane. Cleaning of the membrane is achieved through frequent permeate backpulsing and occasional chemical backwashing. 2) The second configuration is the recirculated (external) MBR, which involves the recirculation of the mixed liquor through a membrane module that is outside the bioreactor. The driving force is the pressure created by high cross-flow velocity along the membrane surface .(Cicek, 2003). ANAEROBIC BIOREACTORS: As the name indicates, these bioreactors are designed to carry out

biodegradation in the absence of oxygen. The anaerobic process comprises a series of interdependent phases. Initially complex organic compounds such as lipids, proteins and carbohydrates, if present, are hydrolyzed to simpler organics. The latter are then fermented to volatile fatty acids (VFAs) by acidogens .The most common of these fatty acids is ethanoic acid. However, propanoic, butanoic and pentanoic acids may also be present in varying quantities depending on the stability of the process. The acidogens include both facultative and obligate anaerobic bacteria. Subsequent to the acidogenic phase is the methanogenic phase. The methanogens are obligate anaerobes and they convert the fatty acids from acidogenesis to methane and carbon dioxide. This results in substantial decrease in the organic content of the wastewater. The methane generated offers an avenue for energy recovery (Buyukgungor, Gurel, 2009).

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AEROBIC BIOREACTORS: The aerobic biodegradation process is represented by the following equation CxHy + O2 + (microorganisms / nutrient ) ---------- H2O + CO2 + biomass. Aerobic treatment of waste is the degradation and purification process in which bacteria that thrive in oxygen rich environments break down and digest the waste. The mixed aerobic microbial consortium uses the organic carbon present in the effluent as their carbon and energy source. The complex organics finally get converted to microbial biomass (sludge) and carbon di oxide (Behera,2009). DIGESTION PATHWAY During the oxidation process, continuous contaminants and pollutants are broken down into end products such as carbon dioxide, water , nitrates, sulphates and biomass (microorganisms). In the aerobic system, the substrate is used as a source of carbon and energy. Synthesis Waste + Oxygen + Microorganisms Respiration Energy + End products More microorganisms

It serves as an electron donor, resulting in bacterial growth. The extent of degradation is correlated with the rate of oxygen consumption in the same substrate. Two enzymes primarily involved in the process are di and mono oxygenases. The latter enzyme can act on both aromatic and aliphatic compounds, while the former can act only on aromatic compounds. Another class of enzymes involved in aerobic condition is peroxidases. TREATMENT OF OIL SPILLS: Petroleum based products are the major source of energy for industry and daily life. Leaks and accidental spills occur regularly during the exploration, production, refining, transport and storage of petroleum and petroleum products. The amount of natural crude oil seepage was estimated to be 600,000 metric tons per year with a range of uncertainty of 200,000 metric tons per year. Release of hydrocarbons into the environment whether accidental or due to human activities is a main cause of water and soil pollution. Basically, the oil in the oily wastewater can be classified into three fractions: free oil, oil/water emulsion and soluble components (Thanh, 2002).

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Biodegradation by natural populations of microorganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants can be removed from the environment and is cheaper than other remediation technologies. The success of oil spill bioremediation depends on ones ability to establish and maintain conditions that favor enhanced oil biodegradation rates in the contaminated environment ( Izanloo, 2007). One important requirement is the presence of microorganisms with the appropriate metabolic capabilities. If these microorganisms are present, then optimal rates of growth and hydrocarbon biodegradation can be sustained by ensuring that adequate concentrations of nutrients and oxygen are present and that the pH is between 6 and 9. The physical and chemical characteristics of the oil and oil surface area are also important determinants of bioremediation success. There are the two main approaches to oil spill bioremediation : (a) Bioaugmentation, in which known oil-degrading bacteria are added to supplement the existing microbial population, and (b) Biostimulation, in which the growth of indigenous oil degraders is stimulated by the addition of nutrients or other growth limiting cosubstrates PLACKETT-BURMAN DESIGN The Plackett-Burman Design was developed by R. L.Plackett and J.P.Burman. Here the design of experiments looks like a matrix, has variables across and runs down. It is used to study the effects of design parameters on the system states so that intelligent design decisions can be made. The design was basically meant to improve the quality control process. It can be used to find out the upper and lower limits of a variable. Through this, the quality of a product can be improved in a less expensive way. Overall quality improvement can save time and money. The study of factors influencing the production of biomolecules is very much essential in any bioprocess development. Generally a higher productivity has been achieved by culture medium optimization. The classical practice of changing one variable at a time while keeping others at a constant level was found inefficient. This single dimensional task does not explain interaction effects among the variables and their effect on the fermentation process. (Aravindan Rajendran,et al,2008).

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Moreover it is a time consuming laborious practice because of the large number of experiments. Conversely, rapid statistical approach enables us to obtain the physicochemical parameters and factors influencing the fermentation process with limited number of planned experiments. One such approach is Plackett-Burman design that allows efficient screening of key variables for further optimization. For the given number of observation the linear effect of all factors can be screened with maximum accuracy. This design is practical when investigating large number of factors to produce optimal or near optimal response.(Aravindan Rajendran,et al,2008).

The cell growth and accumulation of metabolic products in bacteria are strongly influenced by media composition such as carbon sources, nitrogen sources and inorganic salts. Thus it is difficult to search for major factors and to optimize them for biotechnological factors as several factors are involved. The classical method of optimization involves changing one variable at a time by keeping other factors at fixed levels. Statistical method for optimization of media effectively tackles the problem, which involves specific design of experiments which minimizes the the error in determining the effect of variables. Placket-Burman design allows reliable short listing of medium components in fermentation for further optimization and allows one to obtain unbiased estimates of linear effects of all factors with maximum accuracy for given number of observations. (Aravindan Rajendran,et al,2007).

AIM: The present study is focused on the application of Plackett-Burman Design for the comparative study of the efficiencies of membrane bioreactors for the treatment of hydrocarbons.

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OBJECTIVES OF THE WORK:

Design a Membrane bioreactor incorporating both aerobic and anaerobic phases. Formulation of a media that enhances the growth and activity of pseudomonas. Determination of the maximum concentration with which the organism acts. Comparison of the efficiencies of different types of bioreactors. Comparison of the efficiencies of different microorganisms. Measuring the Biochemical Oxygen Demand. Estimation of the Chemical Oxygen Demand.

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MATERIALS AND METHODOLOGY

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MATERIALS SAMPLES TAKEN 1) Diesel 2) Automobile Engine Oil 3) Machine Oil (Heavy Machines) 4) Petrol 5) Lubrication Oil (Small Machines)

TEST MICROORGANISMS

The bacterial strains used in this study were

Pseudomonas Bacillus

CHEMICALS USED 1)FOR PERFORMING PLACKETT- BURMAN DESIGN a) Magnesium Sulphate b) Calcium Chloride c) Di Hydrogen Potassium Phosphate d) Ammonium Nitrate e) Ferric Chloride f) Sodium Chloride g) Glucose h) Sodium Carbonate

2)FOR PERFORMING CHEMICAL OXYGEN DEMAND TEST

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a) POTASSIUM DICHROMATE SOLUTION (0.1 N) b) SODIUM THIOSULPHATE(0.1M) c) SULPHURIC ACID(2M) d) STARCH SOLUTION e) POTASSIUM IODIDE (10%)

3. FOR PERFORMING BIOLOGICAL OXYGEN DEMAND TEST

SODIUMTHIOSULPHATE ( 0.02N) MANGANESE SULPHATE : 48% ALKALINE IODINE: STARCH INDICATOR : 1% CONCENTRATED SULPHURIC ACID.

GLASSWARES USED

FOR PLACKETT-BURMAN DESIGN Boiling tubes and conical flasks

FOR PERFORMING COD Burette, conical flasks, beakers, pipettes etc.

FOR PERFORMING BOD Burette, BOD bottles, conical flasks, beakers, pipettes.

SETTING UP OF BIOREACTORS

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Plastic jars Drip bottles Syringes Drip tubes Aerator Air controller Flow regulator Other requirements: glue, packing tape, M-seal.

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METHODOLOGY

1) BIOREACTOR;

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The membrane bioreactor (MBR) was installed in the Biochemistry laboratory of S.B College, Changanassery. The water sample to be treated during the experiment is prepared by mixing sterilized water and Oils. .

The MBR consisted of cylindrical bioreactors with a working volume of 5 L and 3 L. The bioreactors were made of plastic jars. An outlet is fixed 5 cm above the jars bottom level. Similarly, on the top of each bioreactor jar, a hole was made through which the connection tubes were inserted. The sample water was first stored in the storage tank. From there, the sample was pumped into the constant water level tank. This tank controlled the influent and kept the water level in the bioreactor constant as the inflow rate was set by the water level. The effluent rate of flow was controlled by a flow meter. Aeration pipes were placed in the jars to provide oxygen for the microorganisms and to generate a shear force which hindered membrane fouling. Two membrane bioreactors were used in the experiment, which were operated in a steady state.
a) Aerobic + Anaerobic MBR

((

(Aerobic Tank with Aerator & Agitator)

Flow Regulator (Aerobic Tank With Aerator & Agitator)

Flow Regulator (Tank for Back washing) Relesed to Membrane filter + Flow regulator Effluent

b) Anaerobic + Aerobic Membrane Bioreactor

Anaerobic Tank -1

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Flow Regulator
Anaerobic Tank-2

Flow Regulator

Aerobic Tank with aerator, agitator and membrane

Flow Regulator ( Storage Tank for Back Washing) Flow Regulator

Effluent

2) PROCEDURE FOR PLACKETT-BURMAN DESIGN 1) All the chemicals listed in the table were weighed accurately. 2) 12 tubes were arranged serially and labelled as T1-T12 respectively.

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3) To each tube, the weighed chemicals were added one by one as shown in the table.
4) The chemicals added were properly mixed by little amount of water and the volume in

all the tubes were made up to 30 ml using distilled water. 5) 30ml of distilled water was poured in another tube labelled as 'c' or control. 6) After mixed properly, the tubes were sterilized in the autoclave.
7) The sterilized tubes were taken out, cooled and to each tube 5% oil was added and

mixed well. 8) All the tubes except the control tube 'C' were inoculated with bacilli. 9) The tubes were then incubated at 37 degrees for 3 days.
10) After 3 days, the absorbance of each sample at 440 nm was estimated colorimetrically

and the percentage of degradation of the oil samples was estimated by comparing each with a control.
11) Tube labelled T10 showed a higher efficiency.

T10= 83.33%
12) The concentrations of chemicals added in tube T10 were found to be the most efficient.

Trial

Level and concentration of variable ( g/L ) X1 X2 X3 X4 MgSO4 CaCl2 KH2PO4 NH4NO3

X5 FeCl3

X6 NaCl

X7 Glucose

X8 Na2CO3

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T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12

0.6 0.6 0.2 0.6 0.6 0.6 0.2 0.2 0.2 0.6 0.2 0.2

0.2 0.6 0.6 0.2 0.6 0.6 0.6 0.2 0.2 0.2 0.6 0.2

1 3 1 3 3 1 1 1 3 3 3 3

1 3 1 3 3 1 3 3 3 1 1 1

0.05 0.O5 0.15 0.05 0.15 0.15 0.05 0.15 0.15 0.15 0.05 0.05

1 1 1 3 1 3 3 3 3 3 3 1

3 1 1 1 3 1 3 1 1 3 3 1

0.1 0.3 0.1 0.1 0.1 0.3 0.1 0.3 0.3 0.1 0.3 0.1

3) THE WINKLER METHOD- Measuring Dissolved Oxygen PROCEDURE: a) Carefully fill a 300 ml glass Biological Oxygen Demand stoppered bottle brim- full with sample water. b) Immediately add 2ml of manganese sulphate to the collection bottle by inserting the calibrated pipette just below the surface of the liquid. c) Add 2ml of alkaline potassium iodide reagent in the same manner. d) Stopper the bottle with care to be sure no air is introduced. Mix the sample by inverting several times. Check for air bubbles; discard the sample and start over if any are seen. If oxygen is present, a brownish orange cloud of precipitate or floc will appear. When this floc has settle to the bottom, mix the sample by turning it upside down several times and let it settle again.

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e) Add 2ml of concentrated sulphuric acid via a pipette held just above the surface of the

sample. Carefully stopper and invert several times to dissolve the floc. At this point, the sample is fixed . f) Add 2ml of starch solution, resulting in blue colour. g) Continue slowly titrating until the sample turns clear.
h) The concentration of dissolved oxygen in the sample is equivalent to the number of

milliliters of titrant used.

4) PROCEDURE FOR CHEMICAL OXYGEN DEMAND: a)

50ml of the untreated sample was taken in a dry conical flask.

b) In another conical flask 50ml of distilled water was taken separately. c) To both of the conical flasks 5ml of potassium dichromate was added. These were then kept in boiling water bath for 1 hour at 100 degrees.
d) Samples were then taken out and were cooled. To each conical flask 5ml of potassium

iodide and 10ml of sulphuric acid were added. Mixed well. e) In the mean time a burette was washed, cleaned and was rinsed with sodium thiosulphate solution. Burette was then filled up to the zero mark with sodiumthiosulphate solution. f) The samples in the conical flask were titrated against this sodiumthiosulphate solution to get a pale yellow colored solution. g) At this point 2ml of saturated starch solution was added, mixed and was again titrated until the blue color disappears.
h) This value was note as the COD value of day 1. Similarly the process was repeated after

3 days to get the COD value of day 3. Both the COD values were compared and substituted in the formula to get the percentage of COD change.

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RESULT AND DISCUSSION

1)PLACKETT- BURMAN DESIGN The Optical Density measurement was carried out for the media in all the tubes used in Plackett Burman design and the values obtained are then compared with that of a Control. This shows the percentage of degradation of the oil content in each tube.

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The percentage of degradation is calculated as follows; Percentage of degradation = Optical density of untreated sample - Optical density of treated sample / Optical density of treated sample 100 The Optical Density readings at 440 nm along with percentage of degradation are represented as follows; Test tubes (Serial No) T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 Control (Untreated sample) 0.29 0.04 0.06 0.04 0.05 0.23 0.06 0.05 0.04 0.06 0.05 0.05 Test (Treated sample) 0.22 0.01 0.02 0.01 0.02 0.09 0.02 0.03 0.01 0.01 0.01 0.01 Percentage of degradation 24.14 75 75 75 60 39.01 66.66 60 75 83.33 80 80

The media in the tube labelled as T10 shows the highest rate of degradation. It has increased concentration of Magnesium sulphate, Potassium dihydrogen phosphate, Ferric chloride, Sodium chloride and Glucose. This clearly indicates that the growth and proliferation of Pseudomonas is enhanced by these compounds and hence by increasing the concentration of these compounds, the activity of the organism is enhanced. The Plackett Burman media,T 10, because of its increased efficiency , was selected for the further degradation studies

BOD analysis (Nutrient broth Vs Plackett- Burman Media, T10) Organism: Pseudomonas Day 1 BOD Day 3 D1 ( mg /L) D2 ( mg /L) Percentage 0f decrease D1- D2 /D1 100 48.15

Nutrient

5.4

2.8

18.09

9.38

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Broth PlackettBurman Media, T10 5.4 2.2 18.09 7.37 59.26

D1 - Dissolved Oxygen content of the untreated sample. D2 Dissolved Oxygen content of the treated sample. FORMULA: Dissolved Oxygen content (D) = K 200 0.698 Volume of sodium thiosulphate used Volume of sample Where K is a constant with a value of 1.2.

COD analysis (Nutrient broth Vs Plackett- Burman Media, T3 & T10) Day 1 COD Day 3 Change in COD (x %) 49.23 40 Percentage 0f decrease in COD 100 x 50.77 60

Nutrient Broth Plackett- Burman Media, T10

18.5 18.5

14.5 13.7

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FORMULA FOR CHEMICAL OXYGEN DEMAND: COD = 8 0.1(Volume of sample run- Volume of 50 Change in COD = final day COD/ 1st COD 100 Decrease in COD= 100- Change in COD. The Biochemical Oxygen Demand (BOD) and the Chemical Oxygen Demand (COD) studies were carried out using three mediums; 1)Nutrient Broth Media 2) Plackett- Burman Media,T3 The media T10 shows the highest percentage of reduction, both in BOD and COD. Hence it was selected for the further biodegradation studies . BOD analysis (5 % Diesel) Bioreactor Types

distilled water run)

Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor.

Organism : Pseudomonas Day 1 Bioreactor Type Aerobic + Anaerobic Anaerobic + Aerobic 4.5 1.4 Day 3 D1 ( mg /L) D2 ( mg /L) BOD (mg /L) Percentage 0f decrease D1- D2 /D1 100 68.89

15.08

4.69

10.39

4.5

1.6

15.08

5.36

9.72

64.46

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COD analysis (5 % Diesel) Bioreactor Types

Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor. Organism : Pseudomonas Day 1 Day 3 COD (1st Day) COD (3rd Day) Change in COD (x %) 16.67 25 Percentage 0f decrease in COD 100 x 83.33 75

PARTICULARS

Aerobic + Anaerobic Anaerobic + Aerobic

18.2 18.2

12.2 12.4

0.12 0.02

0.12 0.03

The first experiment was carried out in a sample with 5% Diesel. Samples were collected both before and after treatment. Two types of membrane bioreactors were used; 1) Aerobic + Anaerobic membrane bioreactor. 2) Anaerobic + Aerobic membrane bioreactor. 10 ml of the pseudomonas containing media, T10 was used. The aerobic + anaerobic membrane bioreactor was found to best with reduction in BOD upto 67.26% and COD reduction upto 83.33 % within 3 days. BOD analysis (10 % Diesel) Bioreactor Types

Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor. Day 1 Day 3 D1 ( mg /L) Organism : Pseudomonas D2 ( mg /L) B0D (mg /L) Percentage 0f decrease in dissolved oxygen D1- D2 /D1 100 67.26

Bioreactor Type

Aerobic + Anaerobic Anaerobic + Aerobic

5.5

1.8

18.42

6.03

12.39

5.5

18.42

6.70

11.72

63.63

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COD analysis (10 % Diesel) Bioreactor Types Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor. Day 1 COD Day 3 COD ( 1st Day) COD (3rd Day) Change in COD (x %) 23.1 Percentage 0f decrease in COD 100 x 76.9

Organism : Pseudomonas

Aerobic + Anaerobic Anaerobic + Aerobic

18.6

12.8

0.13

0.03

18.6

13

0.13

0.04

26.92

73.08

The efficiency of the biodegradation was found to be 76.29% for COD and 67.26% for BOD within 3 days. Since a fair percent of degradation was obtained, attention was turned towards the treatment of oil mixture. BOD analysis (5 % Oil mixture) Bioreactor Types

Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor. Day 1 Day 3 D1 ( mg /L) Organism : Pseudomonas D2 ( mg /L) B0D (mg /L) Percentage 0f decrease in dissolved oxygen D1- D2 /D1 100 66.65

Bioreactor Type

Aerobic + Anaerobic Anaerobic + Aerobic

5.1

1.7

17.09

5.70

11.39

5.1

1.9

17.09

6.37

10.72

62.75

COD analysis (5 % Oil mixture )

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Bioreactor Types Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor. Day 1 COD Day 3 COD (1st Day) COD (3 Day)
rd

Organism : Pseudomonas Change in COD (x %) 25.85 29.54 Percentage 0f decrease in COD 100 x 74.15 70.46

Aerobic + Anaerobic Anaerobic + Aerobic

18.5 18.5

12.6 13

0.13 0.13

0.033 0.038

BOD analysis (5 % Oil mixture) Bioreactor Types Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor.

Organism : Bacillus Day 1 Bioreactor Type Day 3 D1 ( mg /L) D2 ( mg /L) B0D (mg /L) Percentage 0f decrease in dissolved oxygen D1- D2 /D1 100 60.08 54.89

Aerobic + Anaerobic Anaerobic + Aerobic

5.1 5.1

2 2.3

17.09 17.09

6.70 7.71

10.39 9.38

COD analysis (5 % Oil mixture )

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Bioreactor Types Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor. Day 1 COD Day 3 COD (1st Day) COD (3rd Day) Change in COD (x %) 34.46 39.23 Percentage 0f decrease in COD 100 x 65.54 60.77

Organism : Bacillus

Aerobic + Anaerobic Anaerobic + Aerobic

18.5 18.5

13.4 13.7

0.13 0.13

0.045 0.051

BOD analysis (10 % Oil mixture) Bioreactor Types Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor.

Organism : Pseudomonas Day 1 Bioreactor Type Day 3 D1 ( mg /L) D2 ( mg /L) B0D (mg /L) Percentage 0f decrease in dissolved oxygen D1- D2 /D1 100 64.98 61.64

Aerobic + Anaerobic Anaerobic + Aerobic

6 6

2.1 2.3

20.10 20.10

7.04 7.71

13.06 12.39

COD analysis (10 % Oil mixture ) Bioreactor Types

P a g e | 36

Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor.

Organism : Pseudomonas Day 1 COD Day 3 COD (1st Day) COD (3rd Day) Change in COD (x %) 30.28% 33.80 Percentage 0f decrease in COD 100 x 69.72 66.20

Aerobic + Anaerobic Anaerobic + Aerobic

19.4 19.4

13.2 13.5

0.1424 0.1424

0.043 0.048

BOD analysis (10 % Oil mixture) Bioreactor Types Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor.

Organism : Bacillus Day 1 Bioreactor Type Day 3 D1 ( mg /L) D2 ( mg /L) B0D (mg /L) Percentage 0f decrease in dissolved oxygen D1- D2 /D1 100 59.95 53.33

Aerobic + Anaerobic Anaerobic + Aerobic

6 6

2.4 2.8

20.10 20.10

8.05 9.38

12.05 10.72

COD analysis (10 % Oil mixture )

P a g e | 37

Bioreactor Types Aerobic + Anaerobic membrane Bioreactor & Anaerobic + Aerobic membrane Bioreactor.

Organism : Bacillus

Day 1 COD

Day 3

COD (1st Day)

COD (3 Day)
rd

Change in COD (x %) 35.71 40

Percentage 0f decrease in COD 100 x 64.29 60

Aerobic + Anaerobic Anaerobic + Aerobic

19.4 19.4

13.7 13.5

0.1424 0.1424

0.05 0.056

The results obtained from the Biochemical Oxygen Demand and the Chemical Oxygen Demand shows the highest efficiency of degradation of the aerobic + anaerobic membrane bioreactor, compared with that of anaerobic + aerobic membrane bioreactor. It shows an efficiency upto 83.33% for COD and BOD reduction upto 68.89%.Pseudomonas was found to be more effective than Bacillus in degrading hydrocarbons.

P a g e | 38

70

Plack - B ett urm Media V Nutrient B an s roth (Pseudom onas)

60

50

40

BOD COD

30

i d D f o g a t n c r e P

20

10

0 Nutrient Broth Plackett Burm Media,T 10 an

B and COD analysisof 5%D OD iesel (Pseudom onas)

90 80 70 60 50 40 30

BOD COD

i d D f o g a t n c r e P

20 10 0 Aerobic +Anaerobic Bireactor Anaerobic +Aerobic Bioreactor

P a g e | 39

B and COD analysisof 10%D OD iesel (Pseudom onas)

90 80 70 60 50 40

BOD COD

i d D f o g a t n c r e P

30 20 10 0 Aerobic +Anaerobic Bireactor Anaerobic +Aerobic Bioreactor

76 74 72 70 68 66 64

B and COD analysisof 5%Oil m OD ixture (Pseudom onas)

BOD COD

i d D f o g a t n c r e P

62 60 58 56 Aerobic +Anaerobic Bireactor Anaerobic +Aerobic Bioreactor

P a g e | 40

68 66 64 62 60 58 56

B and COD analysisof 5%Oil m OD ixture(B acillus)

BOD COD

i d D f o g a t n c r e P

54 52 50 48 Aerobic +Anaerobic Bireactor Anaerobic +Aerobic Bioreactor

72 70 68 66

B and COD analysisof 10%Oil m OD ixture (Pseudom onas)

BOD 64 62 COD

i d D f o g a t n c r e P
60 58 56 Aerobic +Anaerobic Bireactor Anaerobic +Aerobic Bioreactor

P a g e | 41

B and COD analysisof 10%Oil m OD ixture(B acillus)

70

60

50

40

BOD COD

30

i d D f o g a t n c r e P

20

10

0 Aerobic +Anaerobic Bireactor Anaerobic +Aerobic Bioreactor Percentage Of Change In BOD & COD

The efficiency of Bioreactors under different conditions such as variations in treatment phase, concentration of oil content, type of organism used were studied and is tabulated in percentage as follows SERIAL NO. PARTICULARS BOD Aerobic Anaerobic + + Anaerobic Aerobic Bioreactor Bioreactor 68.89 67.26 66.65 60.08 64.98 59.95 64.46 63.63 62.75 54.89 61.64 53.33 COD Aerobic Anaerobic + + Anaerobic Aerobic Bioreactor Bioreactor 83.33 76.9 74.15 65.54 69.72 64.29 75 73.08 70.46 60.77 66.20 60

1 2 3 4 5 6

5 % Diesel (Pseudomonas) 10 % Diesel (Pseudomonas) 5 % oil mixture (Pseudomonas) 5 % oil mixture (Bacillus) 10 % oil mixture (Pseudomonas) 10 % oil mixture (Bacillus)

P a g e | 42

The results clearly indicates that the activity and proliferation of the bacteria, Pseudomonas is greatly increased by the presence of oxygen. After the activation, it can effectively degrade hydrocarbons and use it for energy and other metabolic activities. This is indicated by the tests conducted to measure the Biochemical Oxygen Demand and the Chemical Oxygen Demand. Presence of oxygen is necessary for enhancing the activity of Pseudomonas at the initaial condition.

P a g e | 43

APPENDIX

P a g e | 44

TABLE SHOWING THE CONCENTRATIONS CHEMICALS USED IN PLACKETT-BURMAN DESIGN

1) Trial Level and concentration of variable ( g/L ) X1 X2 X3 X4 MgSO4 CaCl2 KH2PO4 NH4NO3 0.6 0.6 0.2 0.6 0.6 0.6 0.2 0.2 0.2 0.6 0.2 0.2 0.2 0.6 0.6 0.2 0.6 0.6 0.6 0.2 0.2 0.2 0.6 0.2 1 3 1 3 3 1 1 1 3 3 3 3 1 3 1 3 3 1 3 3 3 1 1 1 X5 FeCl3 0.05 0.O5 0.15 0.05 0.15 0.15 0.05 0.15 0.15 0.15 0.05 0.05 X6 NaCl 1 1 1 3 1 3 3 3 3 3 3 1 X7 Glucose 3 1 1 1 3 1 3 1 1 3 3 1

OF

X8 Na2CO3 0.1 0.3 0.1 0.1 0.1 0.3 0.1 0.3 0.3 0.1 0.3 0.1

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12

2)FOR PERFORMING CHEMICAL OXYGEN DEMAND TEST

P a g e | 45

POTASSIUM DICHROMATE SOLUTION (0.1 N): 3.676g of potassium dichromate (K2Cr2O7) in 1L distilled water.

SODIUM THIOSULPHATE(0.1M): 5.811g of sodium thiosulphate (Na2S2O3)in 2L of distilled water.

SULPHURIC ACID(2M): 10.8ml of concentrated sulphuric acid (H2SO4) in 100ml of distilled water

STARCH SOLUTION: 0.5g in 50ml distilled water. POTASSIUM IODIDE (10%): 5 g in 50ml

3. FOR PERFORMING BIOLOGICAL OXYGEN DEMAND TEST SODIUMTHIOSULPHATE ( 0.02N): 0.49g of sodiumthiosulphate in 100ml distilled water. MANGANESE SULPHATE : 48% ALKALINE IODINE: 7.5g of potassium iodide in 70% potassium hydroxide. STARCH INDICATOR : 1% CONCENTRATED SULPHURIC ACID.

P a g e | 46

Conclusion

CONCLUSION

P a g e | 47

In this study a media formulated by Plackett- Burman design,T10 was found as a suitable medium for the growth and activity of pseudomonas. This media was used in the Membrane Bioreactors for the treatment of water samples by pseudomonas.. T10 media provided the entire essential nutrients for the organism so that it can grow and degrade oils efficiently. Under this study a lab scale Membrane bioreactor was designed and was used to compare the efficiency of various aerobic- anaerobic bioreactors in the treatment of oils. A comparative study of the efficiencies of the organisms, Pseudomonas and Bacillus, in the degradation of the hydrocarbon content of the oil samples, under different conditions (Aerobic + Anaerobic and Anaerobic + Aerobic) were carried out. It was found that the aerobic + anaerobic bioreactor was the best with 83.33% reduction in COD and more than 60% reduction in BOD. In both these cases, Pseudomonas, was found to be the best, since it depends the oils for its energy and growth. Hence it can degrade the oil content far faster and better than bacilli and it is indicated accurately by the Biochemical Oxygen Demand Test and the Chemical Oxygen Demand Test.

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P a g e | 49

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