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What is bioremediation?

Bioremediation is a natural approach to cleaning up a contaminated zone with the use of biological agents (such as bacteria or fungi) which are present in the zone, or added to it for this purpose.

How can we demonstrate that the reduction of pollution is caused by the presence of bacteria and not by other factors?

You can perform analyses using biomarkers, (biological markers,) that let you know that the contaminants’ degradation is not due to physical actions such as evaporation or photo-oxidation (structure modification of a compound by sunlight), or to the union of the contaminant with soil components (by adsorption or absorption), but rather the consequence of bacterial activity.

What is a biomarker (biological marker )?

A biological maker is a compound which is part of the contaminant agent and, which, under physical process, has a similar behavior to the product which is to be eliminated. It is not the contaminant and can be the last part of the contaminant to be attacked by biological action (bacteria activity, fungi, etc…)

What is a hydrocarbon?

A hydrocarbon is a compound composed exclusively of carbon and hydrogen atoms joined together to form a chain which can be more or less long depending on the number of carbon atoms that form . This string can be simple or branched, open or closed.

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Schematic representation of the structure of a simple linear hydrocarbon chain. Each vertex is a carbon atom and lines represent connections between them .

How do bacteria break down hydrocarbons?

For some bacteria, hydrocarbons are a source of carbon. Those bacteria break down hydrocarbons, extract the carbon atoms they contain and use them for their growth and reproduction. In this manner, the hydrocarbons located in an area degrade and disappear by turning into new bacteria, CO2 and water.

What are the components of diesel fuel?

Diesel fuel is a petroleum derivative composed of about 75% of linear hydrocarbons and 25% of aromatic hydrocarbons (cyclic) with a length between 9 and 26 carbon atoms. By laboratory techniques such as gas chromatography and mass spectrophotometry, we can identify each hydrocarbon that composes gasoil fuel with graphs, (chromatograms,) see below, in which each peak represents a hydrocarbon with a specific number of carbon atoms. Peak height indicates hydrocarbon quantity.

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¿Cuáles son los biomarcadores en una contaminación por hidrocarburos?

Pristane, phytane and hopane are the markers which can be used for a hydrocarbon contamination such as petroleum or its derivatives spillage (gasoil fuel, oil, etc…)

What are pristane and phytane?

Pristane and phytane are compounds present in petroleum and its derivatives. They are composed entirely of hydrogen and carbon atoms with a branched structure, more complex than a linear one. This branched structure is the reason why bacteria cannot break those substances easily.
On the other hand, pristane and phytane have the same behavior than hydrocarbons of the same length (C17 and C18) under physical effect like evaporation or photo-oxidation.
All those characteristics are the reason why we can use pristane and phytane as biomarkers in bioremediation for petroleum and it derivatives.

 

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Schematic representation of pristane and phytane structure, and of linear hydrocarbons C17 and C18

What are hopanes biomarkers for?

Hopane are molecules composed of carbon and hydrogen atoms. They have a vegetal origin and are part of petroleum and its derivatives. Their structure is much more complex than other hydrocarbons. In their case, their structure is composed by several carbon chains closed and joined together. A consequence of this complexity is that bacteria cannot break those structures and eliminate hopanes; hopanes also have the same behavior than polycyclic hydrocarbons against physical process. This is the reason why hopanes are used as biomarkers in bioremediation of contamination for petroleum and its derivatives.

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Schema: at the top, schematic representation of hopane structure. Below, schematic representation of aromatic polycyclic hydrocarbon (crisene)

Why can we say that bioremediation of petroleum and its derivatives takes place in a determined environment?

Simply by determining how many substances make up petroleum and its derivatives, such as diesel fuel, and by comparing their disappearance with that of the biomarkers.

When the different components of petroleum and its derivatives disappear faster than the biomarkers, we can say that the disappearance is selective and caused by bacteria activity. In that case, bioremediation occurs. On the contrary, if the disappearance is not selective or similar for all petroleum components, including biomarkers, this means that the decrease of the substance is caused by physical process, such as evaporation, photo-oxidation, etc… In that case, there is no bioremediation.

We exposed at the beginning that different components of petroleum and its derivatives can be quantified by mass chromatography and spectrophotometry. Simply by dividing the amounts present in an area of C17 and C18 by a quantity of biomarkers, pristane and phytane respectively, we will obtain an index value ( and/or ). If the value of those indicators decreases with time, this means that C17 and C18 have disappeared more rapidly than biomarkers. In that case, bioremediation occurred in that zone. The following example has been published in Pollution Bulletin 46 (2003) 887-899, with the following graph:

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In that case, the indicators C17/pristane and C18/phytane decrease from a value of 1,1 and 1,3 at the beginning (day 0) to 0,9 and 0,7 at day 40 (figure A and B). This is a clear indication of bioremediation: a physical process would not cause a selective elimination of components. At the end of the test, there is a drastic disappearance of saturated hydrocarbons (figure C). All “peaks” disappear except those corresponding to pristane and phytane: they do not disappear but just decrease in a significant quantity.


 

Can we use any biomarker in any situation?

 No. There are bacteria with a great capacity to use any type of components, including biomarkers (such as pristane and phytane). Those bacteria use biomarkers just when they have no other option. During the first part of the process, those bacteria have a lot of other carbon sources at their disposal in petroleum, that is why they do not use pristane and phytane, which serve as biomarkers.

During the following phase of the process, simpler carbon source get consumed, and bacteria start to use pristane and phytane; at this moment, they cannot be used as biomarkers. This is the reason why we need hopanes with a complex molecular structure to demonstrate that bioremediation is still happening during the final phases of bioremediation.

Example of fuel bioremediation using BIOPULCHER method

During summer 2014, fuel spillage appeared on more than one kilometer along El Cabron beach in the Canary Islands. The origin could be caused by a cargo fuel tank cleaning in open sea rejected to the coast.

Initially, the fuel has been removed by 200 volunteers. But what can we do about the fuel that we cannot remove? How can it be eliminated?

We proposed BIOPULCHER as bioremediation method to remove fuel remains by bacteria activity in zones where it is not possible to remove fuel in a mechanical way. We took some samples of and we treated them with the BIOPULCHER method in la aboratory. Days 7, 14, 22, 36 and 60, we took some samples from the experiment and we analyzed them in order to evaluate the efficiency of our method as a bioremediation system using indicators C17/pristane and C18/phytane. The following chart shows us the results:

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We can see in chart nº1, after 60 days of incubation, that the total quantity of hydrocarbons C10-C40 is reduced by 75%. Furthermore, as we can see in chart nº2, there is a diminution of indicators C17/pristane and C18/phytane, which indicates the effectiveness of bioremediation.

From those results, we can conclude and affirm that the bioremediation method BIOPULCHER is efficient to eliminate the fuel contamination rejected at EL Carbon Beach.

Bibliography

  1. J.E. Ortiz, M.J. García and J.L. Rodríguez Gallego. Biomarcadores y su utilidad en la evaluación de la biodegradación del petróleo. Industria y Minería, 2003 (351): 41-45

 

  1. Roger C. Prince, David L. Elmendorf, James R. Lute, Chang S. Hsu, Copper E. Haith, James D. Senius, Gary J. Dechert, Gregory S. Douglas, and Eric L. Butier. 17a(H),21@(H)-Hopane as a Conserved Internal Marker for Estimatlng the Biodegradation of Crude Oil. Envkon. Scl. Technol. 1004, 28, 142-145

 

  1. Albert D. Venosa, Makram T. Cuidan, Brian A. Wrenn, Kevin L. Strohmeier, John R. Haines, B. Loye Eberhart, Dennis King, and Edith Holder. Bioremediation of an Experimental Oil Spill on the Shoreline of Delaware Bay. Environ. Sci. Technol. 1996, 30, 1764-1775

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