Study on growth kinetics of bacterial

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Environmental concerns are the important aspect behind the process of desulphurization. Diesel is a mixture of many different hydrocarbon molecules and sulfur compounds are the most harmful. Sulfur oxides are generated during burning of the diesel due to presence of sulfur compounds in it. Therefore, the reduction of total sulfur in diesel fuel to an ultra low level is highly necessary to ensure a clean environment.

Most of the sulfur in diesel is converted to sulfur dioxide, but a small amount is also converted to sulfuric acid. In the atmosphere, gaseous sulfur dioxide can also be converted to sulfuric acid and sulfate-containing particles. Thus, atmospheric concentrations of sulfur dioxide are often highly associated with acidic particles, such as sulfuric acid particles. This is the cause of "Acid Rain" and contributes to widespread ecosystem damage. These oxides of sulfur even pollute rivers and lakes, damages buildings, causing serious damage to the eco system and other environmental problems. In addition to this, the sulfur compounds corrode the oil pipelines and different process equipment. Sulfate particulate matter is a significant health concern as well as one of the primary pollutants responsible for impaired visibility. Workers exposed to high concentrations of diesel exhaust may have the symptoms like irritation of the eyes, nose and throat, lightheadedness, heartburn, headache, weakness, numbness and tingling in the extremities. For the people suffering with asthma, even relatively short-term, low-level exposures to sulfur dioxide can result in airway constriction leading to difficulty in breathing and possibly contribute to the severity of an asthmatic attack.

Thus, to ensure safety of the environment, this has been regarded as a very important issue and measures have been taken to manage sulfur concentration in the environment. The process of desulphurization of diesel reduces acid rain problems and other health hazards which are caused due to emission of sulfur compounds.



Removal of sulfur is one of the most costly issues facing industrialized countries worldwide and the petroleum industry and is severely limited inviable economic and environmental options. Sulfur removal is desirable because of :-

  • Environmental regulations mandating decreased sulfur in refined products,
  • an increasing level of sulfur in crude oil processed by refiners and
  • high construction, operating and maintenance costs associated with the existence of sulfur in petroleum.

Desulfurization is also attractive to the crude oil production market, where low-sulfur crude oil commands a premium price over high-sulfur crude oil.


Sulfur is usually the third most abundant element in crude oil, normally accounting for 0.05 to 5%, but up to 14%in heavier oils Most of the sulfur in crude oil is organically bound, mainly in the form of condensed thiophenes, and refiners use expensive physicochemical methods, including hydrodesulfurization to remove sulfur from crude oil These high costs are driving the search for more efficient desulfurization methods, including biodesulfurization . In developing a lower cost biologically based desulfurization alternative, promoting selective metabolism of the sulfur component (attacking the C-S bonds) without simultaneously degrading the nonsulfur (C-C bonds) fuel components in organic sulfur will be the most important consideration.

BDS is a proprietary process based on naturally occurring bacteria that can selectively attack and remove organically bound sulfur from petroleum. Enzymes in the bacteria selectively cleave carbon-sulfur bonds in the presence of oxygen to yield an oxygenated sulfur containing hydrocarbon. Operating at essentially ambient temperatures and atmospheric pressure, BDS is expected to provide significant advantages over conventional hydrodesulfurization technology in achieving sulfur levels below those required by regulatory standards, while being flexible enough to desulfurize a wide range of petroleum streams. BDS is also expected to have value in upgrading crude oil. Since the process is oxidative, the addition of hydrogen is not required, thus avoiding a significant element of conventional desulfurization operating costs, reducing energy consumption and emissions (notably CO2) during refining.

The basic steps of the BDS process are:

  • A slurry is created containing the biocatalyst, additives and high-sulfur petroleum and fed to continuous flow bioreactors.
  • The slurry is continuously pumped from the reactors and the desulfurized oil is separated from the oil/aqueous/biocatalyst output stream.
  • The aqueous phase is further treated to separate out the biocatalyst and water.
  • The sulfur byproduct is captured from the aqueous phase as a water-soluble organosulfur compound and removed from the process.
  • The biocatalyst and water are recycled to the bioreactor and spent biocatalyst is drawn off.

Two types of BDS:

1. Reductive C-S cleavage: Reductive desulfurization requires a reducing equivalent. The role of the reducing equivalent is to reduce DBT to biphenyl, releasing the sulfur as hydrogen sulfide. It should be performed under well controlled sulfate reducing anaerobic conditions. Presently there is no commercially significant anaerobic BDS process because of the difficulties in maintaining anaerobic conditions. However, the advantage of this process is that the absence of oxygen prevents the non-specific oxidation of hydrocarbons to colored, acidic, gum-forming products.

2. Oxidative C-S cleavage: This process removes sulfur from DBT and methyl-DBT in a sulfur specific manner without affecting the carbon skeleton. Thus the fuel value of diesel remains unaffected. Because of its specificity for sulfur atoms and of operation under aerobic conditions, studies on BDS are focused on this technique from last decade. During this process of conversion of DBT to 2-hydroxybiphenyl by Rhodococcus sp. four different molecules are formed. So, this pathway is known as the 4S pathway.


Hydrodesulfurization (HDS) is a catalytic chemical process widely used to remove sulfur (S) from natural gas and from refined petroleum products such as gasoline or petrol, jet fuel, kerosene, diesel fuel, and fuel oils. The purpose of removing the sulfur is to reduce the sulfur dioxide (SO2) emissions that result from using those fuels in automotive vehicles, aircraft, railroad locomotives, ships, gas or oil burning power plants, residential and industrial furnaces, and other forms of fuel combustion.

Another important reason for removing sulfur from the naphtha streams within a petroleum refinery is that sulfur, even in extremely low concentrations, poisons the noble metal catalysts (platinum and rhenium) in the catalytic reforming units that are subsequently used to upgrade the octane rating of the naphtha streams.

The industrial hydrodesulfurization processes include facilities for the capture and removal of the resulting hydrogen sulfide (H2S) gas. In petroleum refineries, the hydrogen sulfide gas is then subsequently converted into byproduct elemental sulfur. In fact, the vast majority of the 64,000,000 metric tons of sulfur produced worldwide in 2005 was byproduct sulfur from refineries and other hydrocarbon processing plants



  1. Peptone (E. Merck)
  2. Beef extract (E. Merck)
  3. NaCl (Ranbaxy)
  4. Distilled Water (Broadway Chemicals)
  5. Diesel - purchased from IOC (Indian Oil Corporation) having sulfur concentration 440 ppm.
  6. Bacterial strain - Rhodococcus sp. (NCIM 2891)

Medium preparation- The medium required for the culturing of Rhodococcus sp. is prepared by mixing peptone, beef extract & sodium chloride (NaCl) in distilled water.

For preparing 1000ml of medium we require the above mentioned substances in following quantities:-


  1. Autoclave - G.B. Enterprise, Kolkata, India.

    Used for sterilization of glass wares and medium.
  2. Hot Air Oven - Bhattacharya & co., Kolkata, India.

    The glasswares are kept in the hot air oven for drying.
  3. Digital Weighing machine - Sartorious.

    Used for measuring weight accurately.
  4. Laminar air flow - Bhattacharya & co., Kolkata, India.

    Used for aseptic transfer of materials to flasks.
  5. BOD incubator cum Shaker - S.C. Dey &co., Kolkata, India.

    After inoculation the flasks are kept in shaker for proper growth of bacteria.
  6. Cooling centrifuge - Remi Instruments Ltd., Mumbai, India.

    After growth of bacteria, the culture is centrifuged to separate the bacterial cell mass which is further dried in hot air oven.
  7. Hot Air Oven - Bhattacharya & co., Kolkata, India.

    The glasswares are kept in the hot air oven for drying.


Dry weight method:-

Determination of cellular dry weight is the most commonly used direct method for determining cell mass concentration and is only applicable for cells grown in solids-free medium. If noncellular solids, such as molasses solids, cellulose or corn steep liquor are present the dry weight measurement will be inaccurate. The samples of culture broth are centrifuged or filtered and washed with a buffer solution or water. The washed wet cell mass is then dried at 80 for 24 hours; then dry cell weight is measured.

Spectrophotometric analysis:-

This method is based on the absorbtion of light by suspended cells in sample culture media. The intensity of the transmitted light is measured using a spectrometer. Turbidity or optical density measurement of the culture medium provides a fast, inexpensive and simple method of estimating cell density in the absence of other solids or light absorbing compounds. The extent of light transmission in a sample chamber is a function of cell density and the thickness of the chamber. Light transmission is modulated both by absorption and scattering. Pigmented cells give different results than unpigmented ones. Background absorption by components in the medium must be considered, particularly if absorbing dissolved species are taken into cells. The medium should be essentially particle free. Proper procedure entails using a wavelength minimized absorption by medium components, blanking against medium, and the use of a calibration curve. The calibration curve relates optical density (OD) to dry weight measurements. Such calibrations can become nonlinear at a high OD values and depend to some extent on the physiological state of the cells.


Growth pattern and kinetics in batch culture à

Growth is a result of both replication and changes in cell size. Microorganisms can grow under a variety of physical, chemical and nutritional conditions. In a suitable medium, organisms extract nutrients from the medium and convert them into biological compounds. Part of these nutrients are used for energy production and part are used for biosynthesis and product formation. As a result of nutrient utilization, microbial mass increases with time and can be described simply

Microbial growth is a good example of an autocatalytic reaction. The rate of growth is directly related to cell concentration, and cellular reproduction is the normal outcome of this reaction.

When a liquid nutrient medium is inoculated with a seed culture, the organisms selectively take up dissolved nutrients from the medium and convert them into biomass. A typical batch growth curve includes the following phases:-

Lag phase - the lag phase occurs immediately after inoculation and is a period of adaptation of cells to a new environment. Microorganisms reorganize their molecular constituents when they are transferred to a new medium. Depending on the composition of nutrients, new enzymes are synthesized, the synthesis of some other enzymes are repressed, and the internal machinery of cells is adapted to the new environmental conditions. These changes reflect the intracellular mechanisms for the regulation of the metabolic processes. During this phase, cell mass may increase a little, without an increase in cell number density.

Logarithmic or exponential growth phase - In logarithmic or exponential growth phase, the cells have adjusted to their new environment. After this adaptation period, cells can multiply rapidly, and cell mass and cell number density increase exponentially with time. This is a period of balanced growth in which all components of a cell grow with the same rate. That is, the average composition of a single cell remains approximately constant during this phase of growth. During balanced growth, the specific growth rate determined from either cell number or cell mass would be the same. Since the nutrient concentrations are large in this phase, the growth rate is independent of nutrient concentration in this phase.

Deceleration phase - The deceleration growth phase follows the exponential phase. In this phase, growth decelerates due to either depletion of one or more essential nutrients or the accumulation of toxic by-products of growth. For a typical bacterial culture, these changes occur over a very short period of time. The rapidly changing environment results in unbalanced growth. In the exponential phase, the cellular metabolic control system is said to achieve maximum rates of reproduction. In the deceleration phase, the stresses induced by nutrient depletion or waste accumulation cause a restructuring of the cell to increase the prospects of cellular survival in a hostile environment.

Stationary phase - This phase starts at the end of deceleration phase, when the net growth rate is zero (no cell division) or when the growth rate is equal to the death rate. Even though the net growth rate is zero during the stationary phase, cells are still metabolically active and produce secondary metabolites. Primary metabolites are growth related products and secondary metabolites are non growth related. In this phase the production of certain metabolites is enhanced due to metabolic deregulation.

During this phase, the cell catabolizes cellular reserves for new building blocks and for energy producing monomers know as endogenous metabolism. The reason for termination of growth may be either exhaustion of an essential nutrient or accumulation of toxic products.

Death phase - the stage following the stationary phase is the death phase. However, some cell death may start during the stationary phase and a clear demarcation between these two phases is not always possible. Often, dead cells lyse, and intracellular nutrients released into the medium are used by the living organisms during stationary phase. At the end of the stationary phase, either because of nutrient depletion or toxic product accumulation, the death phase begins. During the death phase the cells may or may not lyse, and the reestablishment of the culture may be possible in the early death phase if cells are transferred into a nutrient rich medium.

Monod model:-

The relationship of specific growth rate to substrate concentration often assumes the form of saturation kinetics. A single chemical species is assumed, S, is growth rate limiting, (that is, an increase in S influences growth rate, while changes in other nutrient concentrations have no effect). These kinetics are similar to the Langmuir-Hinshelwood kinetics in traditional chemical kinetics or Michaelis - Menten kinetics for enzyme reactions. When applied to cellular systems, these kinetics can be described by the Monod Equation:

where µm is the maximum growth rate when S>>Ks. The constant Ks is known as the saturation constant or half velocity constant and is equal to the concentration of the rate limiting substrate when the specific rate of growth is equal to one-half of the maximum. That is, Ks =S when µ = 1/2µmax. In general, µ = µm for S>>Ks and µ = (µm/Ks)S for S<<Ks. The Monod equation is semiempirical; it derives from the premise that a single enzyme system with Michaelis - Menten kinetics is responsible for uptake of S, and the amount of that enzyme or its catalytic activity is sufficiently low to be growth rate limiting.

The Monod equation describes substrate specific growth only when growth is slow and population density is low.


Batch study for the growth kinetics of Rhodococcus sp. was carried out for 48 hrs in aqueous medium (100%) and the results obtained are given in table1.


The growth kinetics of rhodococcus sp is observed in 100% aqueous medium by performing batch culture. This growth pattern is obtained by plotting a graph of biomass concentration against time. A classical growth pattern is obtained having lag phase, log or exponential phase, deceleration phase, stationary phase and death phase.

Then the bacterium has also been cultured in 90% aqueous medium + 10% diesel (non-aqueous) & 80% aqueous medium + 20% diesel. The biomass concentration in these two mediums was found to be less than the biomass concentration in 100% aqueous medium which shows that, the growth decreases as the substrate concentration is increased.

The bacterium then gets adapted to the non-aqueous medium (diesel) and starts taking sulfur as its nutrient from diesel and as a consequence the sulfur level of diesel is also decreased. Thus, this process can be carried out for desulfurization of diesel thereby reducing sulfur concentration of the environment.