Bioremediation of the Environment Using Oil-Eating Bacteria

Shenade Annie Kerketta, Amity University Kolkata

In nature, certain microbes such as bacteria and fungi can utilize hydrocarbons and other toxic pollutants to carry out metabolic and reproductory processes. For these hydrocarbon-degrading microbes, pollutants are nothing more than another kind of food they can consume and metabolize for energy. These microorganisms can be thought of as nature’s army of microscopic scavengers, cleaning the earth and remediating the ecosystems in an eco-friendly manner. 

Crude oil is one of the most extensively required materials that the human race is using for its rapid modernization and urbanization. And such widespread use increases the chances of accidents such as leaks and spills, which pollute the ecosystem. In the search for environment-friendly solutions to such problems, scientists have discovered novel ways through which these unique microbes could be utilized effectively to remediate such disasters. Years of experimentations performed to study such microbes have revealed which kinds of by-products are desirable for the benefit of the environment and in turn, us humans. Thus giving rise to important processes like biostimulation and bioaugmentation. 

In this article, we’ll be understanding the concept and importance of bioremediation, hydrocarbon-degrading microbes, and how they both come together to reduce various pollution-causing events and accidents. There are microbes that on exposure to crude oil and pollutants secrete enzymes that break down toxic compounds into molecules of water and other harmless gases like carbon dioxide. One of the molecules produced in that process that helps in degrading the crude oil is called a biosurfactant. And through this phenomenon, these microbes execute bioremediation.

Introduction

Bioremediation can be defined as a process in which living organisms such as microbes and bacteria are employed in the eradication of pollutants, contaminants, and toxins from water bodies and terrestrial bodies. It is commonly used for cleaning up oil spills or polluted groundwater. Bioremediation works by stimulating the growth of desirable microbial species such as Pseudomonas sp. and Bacillus sp., which utilize contaminants and pollutants for their growth and survival.

The idea of bioremediation was first proposed by George M. Robinson in the 1960s, and the first-ever large-scale oil spill clean-up took place in 1968. Some of the most notable bioremediation successes are as follows- Exxon Valdez Oil Spill in Alaska in 1989; sewage effluent, Cape Cod Massachusetts; chlorinated solvents, New Jersey; pesticides, San Francisco Bay Estuary; agricultural chemicals in the midcontinent, Gasoline contamination, Galloway, New Jersey.

Ways like using booms, skimmers, sorbents, vacuums, electrocoagulation, electrolytic oxidation, membrane filtration, and shovels are available for oil spill clean-ups. Yet bioremediation proves out to be better because of the cost-effective element and also that it does not harm nature.
Oil spills occur due to natural seeps, oil tankers, offshore oil platforms, and pipeline leaks. These oil spills cause serious damage to the immediate environment, affecting all the animals in that environment, and indirectly, us humans as well. Oil may enter the fur of mammals, it may cover the feathers of birds impairing their flight, it can reduce the dissolved oxygen level killing the marine life, and it can contaminate the drinking water as well. There are many volatile compounds like benzene, ethylene glycol, formaldehyde, methylene chloride, tetrachloroethylene, toluene, xylene, and 1,3-butadiene which are released from these oils, that lead to major air pollution. Due to such alarming widespread consequences, science proposed bioremediation as the solution best suited for the need of the hour.

Knowledge from Initial Researches:

From the initial researches, it was found that microbial species like Pseudomonas, Bacillus, Vibrio, and Flavobacterium degrade petroleum hydrocarbons quite efficiently. Species such as these work by metabolizing or eating petroleum hydrocarbons. They break the petroleum hydrocarbons into smaller carbon molecules, carbon dioxide, and water and derive energy from them for their growth. By degrading the petroleum hydrocarbons they give rise to furthermore microbes which can degrade the oil.

Different microbes attack different petroleum hydrocarbon molecules, for instance, some microbial species degrade saturated hydrocarbons but cannot degrade aromatic hydrocarbons. Alkanes are the easiest to degrade whereas polycyclic aromatic hydrocarbons are the most difficult to degrade for the majority of these species. Microbes use oxygen for bioremediation which is why it is termed as aerobic bioremediation of petroleum hydrocarbons.

One study states that microbes have adapted to degrade aliphatic hydrocarbons which show resistance to degradation due to their insolubility and hydrophobicity. Firstly the oil surface is treated with biological surfactants to break the hydrocarbons into molecules of smaller size than the microbial cells. The surfactants engulf those small hydrocarbon molecules. Now, the microbial cells change their cell membrane lipid composition such that it easily breaks the hydrocarbon droplet. In yeasts, it was found that pore formation takes place in the cell membrane to allow entry of the hydrocarbons. The metabolism of alkanes, branched-chain alkanes, and alkenes has been extensively studied. OCT plasmid was found to play a very vital role in understanding the degradation of aliphatic hydrocarbons. Some of the older papers state the possibility of anaerobic degradation of oils also.

Initial researches eventually discovered polycyclic aromatic hydrocarbon-degrading bacteria. Some of them are as follows Pseudomonas, Arthrobacter, Acinetobacter, Flavobacterium, Alcaligenes, Micrococcus, and Corynebacterium. To isolate these species, methods like DNA hybridization using NAH7-derived gene probes, PAH spray plate assays, 14C-PAH mineralization assays, and dioxygenase activity assays have been used. 

A real-time polymerase chain reaction method had been devised to monitor the natural attenuation of a catabolic gene from anaerobic hydrocarbon-degrading bacteria. The initial researches educate us about the different methods by which petroleum hydrocarbon-degrading bacteria can be isolated, how these bacteria work on the petroleum hydrocarbons (mechanisms), at what conditions they work efficiently, the differences in mechanisms for aerobic and anaerobic degradation methods, what effects will take place if those conditions are to be altered, and the physiology and metabolism of the different hydrocarbon-degrading bacteria.

The main ideology behind the isolation of petroleum hydrocarbon-degrading bacteria is to first take an oil-contaminated sample (liquid or soil) then treat it with mineral salt medium and crude oil. Crude oil exists as the only energy source. After several subcultures, the microbial colonies which show affirmative growth on the crude oil substrate are selected as the oil-degrading microbes. These microbes are then used for further research like characterizing genes responsible for bestowing its oil-degrading or metabolizing capabilities, and finding out which hydrocarbon degrades most easily and under what conditions.

Characterization is often done by examining the physical properties and running systematic analysis based on physiological and biochemical characteristics that were originally recorded in Bergey’s Manual for Determinative Bacteriology and related studies. The 16S rDNA sequence analysis is extensively used for the taxonomic characterization of the isolated species.

Oil-degrading bacterias also degrade substances like pyrene which are present with the oil pollutants. The gist of initial researches is that in nature, there are oil-degrading bacteria that normally degrade the oils secreted by animals. This property has been put in use by humans majorly to clean up oil spills, sewage pipelines, and petroleum-contaminated soil. These oil-degrading microbial species were put to use because researchers found out that the microbes can metabolize the petroleum, harmful elements like petroleum hydrocarbons. These microbes secrete enzymes that metabolize the petroleum hydrocarbon molecules breaking them into smaller carbon molecules, carbon dioxide, water, and energy. The energy is used to generate more microbes. In this way, they are degrading the hydrocarbons and helping us in cleaning toxic oil spills. This knowledge has helped largely in cleaning the oil spills like the Gulf War oil spill, Deepwater Horizon oil spill, Atlantic Empress / Aegean Captain oil leak.

Bioremediation of Water Bodies:

The basic process of bioremediation of water bodies or sewage pipes is to stimulate the growth of already existing hydrocarbon-degrading microbes. These microbes exist in every environment. The process of stimulation of the already existing hydrocarbon-degrading bacteria is called biostimulation. Here, specific nutrients are provided in highly accurate concentrations to maximize the growth of only the desired microbes hence speeding the whole bioremediation process. Some of the major nutrients that are added are nitrogen, phosphorus, nitrogen-rich organic substrates (sugarcane bagasse, banana skin, and rice husk), oxygen, and biosurfactants. When these nutrients have been added the growth of the oil-degrading microbes will increase in population. They will convert the toxic hydrocarbons into alcohol which will be oxidized to an aldehyde and an acid. The acid will be eventually be converted into water thereby cleaning the toxicity. The biostimulants are added during low tide at the high tide line.

The addition of hydrogen degrading microbes as a biostimulant in the bioremediation process is called bioaugmentation. This process is selected as a last resort because it poses many negative effects. In many cases the microorganisms added, compete with the already existing microbes which negate the bioremediation. Bioaugmentation is applied to cases where the existing microbes are unable to degrade the present pollutants.
The factors affecting bioremediation include the energy of the water, the surface area of oil, temperature, oxygen, nutrient content, oil composition, and pH. According to an experiment carried out by the optimum temperature at which degradation of hydrocarbons works best is 20⁰ C. The larger the surface area of the oil spill more is the amount of oil-degrading microbes. Biosurfactants help in breaking the oil molecules into small droplets thereby increasing the surface area and making it easier for the microbes to break the hydrocarbons. The energy of water also plays a vital role, if the waters are rough the degradation becomes difficult because the nutrients get diluted and the contamination spreads. The nutrients, if added in the right proportions leads to an efficient degradation process. If the oil composition is complex the process of degradation becomes difficult.

Biosurfactants

Oil degrading microorganisms secrete molecules called biosurfactants. These molecules are amphiphilic, meaning it has both hydrophobic and hydrophilic parts. These molecules break the oil lumps into smaller droplets and make them available for the microbes so that they can break the hydrocarbons thereby easing up the degradation process. It can be understood like when humans make small proportions of food to fit their mouth, here the hand which is tearing the food or the teeth which bite into the food can be treated as the biosurfactant, the food is the oil to be degraded and the humans are the oil-degrading microbes. The biting or tearing of food by the hand makes the breaking of food/consumption of food easy. The same is the case for these microbes, producing biosurfactant molecules to degrade oil molecules.

Soil Bioremediation

All this while, we were talking about water body bioremediation. Terrestrial bodies also get contaminated by crude oil and its clean-up using the oil-degrading microbes is termed soil bioremediation.

Examples of oil-degrading microbes found in oil-contaminated soil are Micrococci sp., Bacillus sp., Pseudomonas sp., and Staphylococcus sp.

It uses the oil-degrading microbes which are already present in that environment. Biostimulation takes place by adding amendments (vegetable oil, molasses, and baking soda). The existing microbes are stimulated to increase their population and they break down the crude oil contaminants into simpler consumable compounds, carbon dioxide, and water.

Soil bioremediation can take place in two ways- in-situ and ex-situ. In situ methods require digging up of wells to reach the groundwater. The groundwater is treated with nutrients so that the microbes dwelling in its increase in population. After the groundwater is treated and a subsequent amount of microbial population exists then bioremediation takes place at a suitable rate.

The amendments added also include nitrogen, phosphorous, and oxygen. The problem with in situ method is that it is not applicable when the soil density is high (the nutrients will not be able to disperse as needed) and contaminant toxicity is high.

The ex-situ method requires digging up the contaminated soil and place the soil in tanks where it is stirred, heated, and mixed with the nutrients. The microbes increase in population and start degrading the pollutants. Here the pumped-up groundwater treated with nutrients can also be used as a biostimulant. Even for soil remediation factors like temperature, soil moisture, pH, humidity, nutrient availability must be optimum.

Some soil bioremediation projects include the remediation of Borhola oil fields, Assam, India; park of nations in the city of Lisbon; a gas plant in Cannes; microbiological remediation of soil contaminated with thermo oil in Extremadura, Spain.

The general drawbacks of soil remediation are, it is a slow process, laborious process, not all compounds are biodegradable. Despite these drawbacks, bioremediation has proven to be the best method because it is environment-friendly and natural. Scientists are continuously working on ways to speed up the process and decrease the drawbacks. 

Recent advances

From the recent studies on hydrocarbon-degrading microbes, we have found out the following:

• These microbial colonies when mixed in a consortium have a synergistic effect. The consortium gives better results of degradation compared to individual strain results.
• Biochar plays an important role in increasing the efficiency of oil-degrading microbes. So biochar can be used as a biostimulant in soil bioremediation.
• These microorganisms secrete biosurfactant molecules which help in the degradation process (described earlier in this article), these biosurfactant molecules can be isolated and cultured to be used for oil recovery in reservoirs.
• Achromobacter, Azospirillum, and Pseudomonas species are known to best remediate fuel and they work best in a cooperative relationship.
• It is found that these species work best when in a consortium because some species cannot secrete proper biosurfactant amounts so they utilize the biosurfactant secreted from the other species present.
• Stenotrophomonas chelatiphaga and its strains, and new strains isolated from the coastal zones of the Caspian Sea have shown the highest emulsification index, hydrophobicity, and highest value for decreasing oil viscosity. The other new strains found are Sphingobacterium kitahiroshimense and Achromobacter sp. Genetically modified organisms made from the wild type or native strains are the new topic of research in this aspect. They can be used to stimulate microbes and help in oil degradation. Such organisms have been known to degrade hydrocarbons on their own to some extent.
• If proper and accurate biostimulation and bioaugmentation are provided then microbes can degrade oil in freezing environments too.
• Every day new strains of oil-degrading microbes with increasing efficiency are being discovered and researched.
• New methods are being developed to increase the efficiency of bioremediation.
• Pseudomonas sp. is found to be one of the most efficient in bioremediation, when these microbes work together in a cooperative consortium they have been shown to work better and faster.

Conclusion

Bioremediation is an efficient, environment-friendly, natural way of cleaning up organic compounds which pose threat to the environment. This technique can make it possible for us to become less dependant on remediations that utilize harsh chemicals and other non-environment friendly means that also require massive expenditures. It is of utmost importance that scientists of this field are given proper infrastructural and financial support from companies and governments so that the R&D of bioremediation-based techniques is improved.
The process may be slow for now but scientists are constantly developing and improving upon the methods to speed up the process. Ongoing studies have found new abilities of these microbes like their emulsifying ability and their synergistic effect. There have many successes proving that bioremediation is one of our ways to a greener Earth.

Also read: Dispelling Myth and redirecting Facts: Diabetes

Sources

1. Dayamrita, K. K., Divya, K. K., Sreelakshmi, R., Arjun, E. J., & John, F. (2020). Isolation and characterization of hydrocarbon degrading bacteria from oil contaminated soil – Potential for biosurfactant assisted bioremediation. 020009. https://doi.org/10.1063/5.0017395   
2. Geetha, S. J., Joshi, S. J., & Kathrotiya, S. (2013). Isolation and characterization of hydrocarbon degrading bacterial isolate from oil contaminated sites. APCBEE Procedia, 5, 237–241. https://doi.org/10.1016/j.apcbee.2013.05.041
3. Lücking, R., Leavitt, S. D., & Hawksworth, D. L. (2021). Species in lichen-forming fungi: Balancing between conceptual and practical considerations, and between phenotype and phylogenomics. Fungal Diversity. https://doi.org/10.1007/s13225-021-00477-7
4. Tian, X., Wang, X., Peng, S., Wang, Z., Zhou, R., & Tian, H. (2018). Isolation, screening, and crude oil degradation characteristics of hydrocarbons-degrading bacteria for treatment of oily wastewater. Water Science and Technology, 78(12), 2626–2638. https://doi.org/10.2166/wst.2019.025

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