Hydrocarbon Degrading Bacteria: A Remedy for Oil Spills

Some of the famous oil spill disasters were of Alaska particular in Prince William Sound where 200,000 barrels of crude oil has been expanded on sea due to oil spills. In 1946, Claude E. Zobell reviewed the action of 298 microorganisms on hydrocarbons. The activity of microbes depends upon the chemical nature of the compound, and many microbes use hydrocarbons as a sole source of carbon. In 1986, the Tory Canyon named ship sank in the English Channel (Ronald M. Atlas, 1991). This drew the attention of various researchers and environmentalists and accelerated the research on hydrocarbon degradation. Afterward, various researchers have done to analyzed the microbial activity to degrade hydrocarbons so these microbes can be used as a bioremediation agent. Some of the important researchers are studied from published research papers and research journals, and are described below. 

The most successful method for hydrocarbon degradation is through bio-remediation, i.e. use of microbial activity for degrading specific hydrocarbons. Various rate-limiting elements are responsible for microbial activity, i.e., nitrogen (fixed form by using fertilizers), oxygen (molecular form by using Hydrogen Peroxide), and phosphorus (phosphate form), which are given from outside to enhance microbial degradation activity (Ronald M. Atlas, 1991). There are up to 10% hydrocarbon-degrading microbes present in oil-polluted sites of the total microbial community in oil-polluted sites, compared to only 1% presence of hydrocarbon-degrading microorganisms in unpolluted sites. Temperature has a major effect on degradation activity, as decreasing temperature, viscosity increases, whereas volatile activity decreases which resulting in a reduction in degradation activity. The optimum temperature range for the microbial degradation of hydrocarbons is 30-40 degrees Celsius (Ronald M. Atlas, 1991). 

OHCB (Obligate Hydrocarbonoclastic Bacteria) is one of the marine hydrocarbon-degrading bacteria that can grow in high salinity levels, i.e,. They degrade PAH (polyaromatic hydrocarbon) oil spills in the sea. Increasing concentration of oil spills in oceans leads to the creation of an anoxic environment, which is a condition in which water has a scarce amount of dissolved oxygen, which is generally less than 0.5 mg/liter (Yakimov et al.). These OHCB microbes degrade only 2 substrates out of 95 substrates according to BIOLOG Anomaly. These two substrates are Tween 20 and Tween 4,0, which contain long chains of hydrocarbon, particularly alkane (Yakimov et al.). Bio-mitigation studies of various marine hydrocarbon-clastic bacteria (OHCB) are conducted through genomic analysis using 16S rRNA sequencing. For example, Marinobacter could easily degrade hydrocarbons than Alcanivorax as it contains an extra 1500 genes for different enzymes responsible for the degradation of several hydrocarbons by using them as a sole source of carbon (Yakimov et al.). 

Use of single hydrocarbon-degrading bacteria does not fulfill the requirement, so a consortium ( a mixture of microorganisms) is made to overcome the problem of the lower rate of biodegradation of hydrocarbons or crude oil. Farinazaleen et al. observed that consortium 2 (containing 6 strains) degrades crude oil more effectively than consortium 1 (containing 3 strains). Further stated that consortium 2 wholly consists of the Bacillus and Pseudomonas genera species. Their study was performed on microbial degradation of hydrocarbons in the soil for 60 days at room temperature, monitored at an interval of 15 days (Farinazaleen et al.). Moreover, the study revealed that Pseudomonas and Micrococcus were more efficient in degrading hydrocarbons than any other genus. For more studies, the soil was contaminated by diesel oil and engine oil containing different aliphatic compounds and some aromatic compounds. Alkane was degraded by 57% of its original mass by using Consortium 2 and only 42% of total mass by using Consortium 1. Inoculation of basal media containing inorganic salts results in an increase in the number of microbes up to 10 times in 45 days, and then the population starts declining due to an upset in the ratio of inorganic compounds to organic compounds (Farinazaleen et al.). Thomas et al. reported that for the ideal growth of hydrocarbon-degrading microorganisms, the ratio of inorganic compounds to organic compounds must be optimum, i.e. C:N:P ratio should be 120:20:1 (where C - Carbon, N - Nitrogen, and P - Phosphorus) (Thomas et al., 1992).

The petrochemical industry is a serious concern in terms of environmental pollution as it is one of the fastest-growing and economically important industries in Nigeria. But refinery wastewater causes cancerous effects on human health and deleterious effects on indigenous flora and fauna. Usman D. Hamza et al.'s study revealed that microbial degradation of industrial wastewater helps in decontamination of refinery wastewater before releasing it into the environment. Further reported that Micrococcus shows growth of great vertical extent when crude oil is used as a sole source of carbon (optimum pH for growth is 7.1), inorganic and organic matter added up to the optimum condition for enhanced growth (Usman D. Hamza et al.). Microbes were isolated from refinery wastewater using the standard dilution method and grown on a nutrient agar plate, which allows separate colonies. These colonies were further sub-cultured using nutrient agar at 37 degrees Celsius for 48 hours, which were later morphologically and chemically characterized as Serratia marcescens, Bacillus subtilis, Bacillus cereus, and Micrococcus. Both Bacillus subtilis and Micrococcus showed the greater potential of degrading hydrocarbon in wastewater (reduction in COD up to 56% by Micrococcus and value of OD observed 0.9) (Usman D. Hamza et al.).

There are a large number of oil spills occur regularly in the sea, as all bacterial species cannot survive in very high salt concentration so a limited number of halophiles and halotolerant bacteria grow there which use hydrocarbon as a sole source of carbon and energy are generally known as obligate marine hydrocarbon degrading bacteria (OHCB). Guang Guo et al. studied halo-tolerant catechol 2, 3-dioxygenase. They cloned, expressed, and purified two enzymes (catechol 2, 3 3-dioxygenase) from high salinity tolerant microbes and analyzed the effect of salt concentration on the enzymatic activity. The consortium prepared by them degraded 20% NaCl w/v successfully in four days (Guang et al.). They identified microbe using 16s rRNA sequencing and identified Thalassorpira, Nitratireducto, Fodinicurvata, Tilia, Rhodobium, Marispitullum, Pelagibius, etc (Guang et al.). The enzyme showed activity even after complete degradation of phenanthrene, which is most likely due to the formation of catechol in intermediate steps. They inserted C2301 and C2302 genes in E. coli BL21, and the molecular mass of the protein identified after overcoming was 35 KDa (Guang Guo et al.). The enzymes were highly active at 10% NaCl and were also capable of degrading up to 30% NaCl, which makes the halo-tolerant not hemophilic (Guang Guo et al.).

Maliji and Olama et al. studied a total of seven factors that were affecting the hydrocarbon-degrading activity of an enzyme using a statistical design called Plackett-Burman. With the use of the immobilization technique, B. cereus A can be used for the large-scale degradation of hydrocarbons. They collected samples from South Lebanese Saida Port and identified bacterial strains using 16s rRNA sequencing, and incubation was done at 30 degrees Celsius for 48 hours (Maliji and Olama et al.). The screening was done using seawater nutrient agar media containing diesel or oil used as a sole source of carbon and energy (Maliji and Olama et al.). B. cereus A and B showed degradation up to more than 82% and 81%, respectively, at pH 7 and 2% inoculum in 100 mL culture. Extreme pH leads to a negative effect on microbial activity to degrade hydrocarbon, and inoculum more than 2% results in the same effects (Maliji and Olama et al.). Nitrogen, phosphorus, and yeast extract showed a positive effect on microbial growth. B. cereus A adsorbed on luffa showed 80.67% for aliphatic and 78.8% for aromatic degradation, which was significantly higher than B. cereus B (Maliji and Olama et al.). 

Organic compounds, mainly comprising hydrocarbons, are in geological formations deep inside the Earth in the form of crude oil and natural gas. Based on their molecular weight, 13 hydrocarbons can be classified into alkanes, naphthenes, aromatics, and alkenes (Scholz et. al., 1999). Petroleum is the major energy source worldwide, and because of its important position in the world economy, large amounts of toxins are annually released into the environment by seepage. (Ojumu, 2004).