CH391L/S14/Bioremediation&caffeinatedcoli

Bioremediation
Bioremediation is a process that uses living systems, such as microorganisms or plants, to biodegrade or neutralize pollutants in situ or ex situ. In situ bioremediation treats the pollutants on site, while ex situ involves moving the pollutants elsewhere such as into a bioreactor for treatment. Bioremediation has been used to treat heavy metal pollution or organic wastes, such as mercury ions or petroleum byproducts from industry. Many different processes have been developed to make the bioremediation processes more effective and efficient. This can be done by using the indigenous organisms or introducing exogenous species. Some most common methods are as follows:



Bioattenuation
Bioattenuation is the natural way to degrade the pollutants with indigenous microorganisms. Pollutants are transformed into less harmful chemicals by biodegradation or naturally-occurred chemical reactions that stimulated by microorganisms. The time required for bioattenuation depends on the type of pollutants and site conditions. Bioattenuation is more cost-effective than other clean-up technologies #Mulligan2004, since there is only minimum costs for treatments and post clean-up. However, bioattenuation is not available when the site is oligotrophic or lack appropriate microorganisms #Megharaj2011.

Bioaugmentation/Biostimulation
Bioaugmentation and biostimulation are two different approaches that applicable when the indigenous microorganisms are not available or effective for bioremediation. Biostimulation is a way to modify the site condition by adding nutrients to stimulate more bacteria for bioremediation. Many different fertilizers are used for this purpose, such as water-soluble nitrate or phosphate. However, the stoichiometry of nutrients should be taken into concern when applying biostimulation, since many researches showed the change of nitrate or phosphate ratios can also alter the composition of bacterial community, thus affect the efficiency of bioremediation #smith2002#Megharaj2011.

Bioaugmentation is another technique to improve the efficiency of bioremediation by introducing pre-adapted microorganisms. These selected catabolic bacteria were inoculated in the soil that extracted from the contaminated site to improve their persistence and efficiency of biodegradation. The microorganisms are also pre-treated with the pollutant to enhance its metabolic capacity. The disadvantage of bioaugmentation is the lesser competitiveness relative with indigenous species. Per-adapted strains do not always survive well in the contaminated site as they do in the lab. #Blasco1995#Megharaj2011

Phytoremediation
Phytoremediation is the treatment that uses plants for bioremediation. In many cases, microorganisms are also involved in the treatment to increase the efficiency and biocompatibility of the plants. The advantages of phytoremediation are the treatment can reach the deeper soil by the roots, and it can preserve the environment in its natural state (compared with introducing exogenous microorganisms, which can alter the composition of bacterial community). However, it usually takes much longer to finish the treatment, and the pollutants, such as heavy metals, can accumulate in the plants and return to the food-chain #Megharaj2011.



Genetic engineering of microorganisms
Scientists can use genetic engineering to enhance the efficiency of bacteria for biodegradation, or to create new strains for specific treatments. This can be done by either amplifying an existing metabolic enzyme, minimizing the side reactions or pathway bottlenecks, or introducing a heterologous gene to gain new characteristics. The main drawback of genetic engineering microorganisms (GEMs) is the risk of releasing genetic elements to undesired organisms, such as pathogens. Many techniques are used to ensure the safety of GEMs, such as a programmed mechanism to trigger cell death after treatment is completed #Pandey2005. However, few GEMs are allowed to use for field application due to strict law restriction. Pseudomonas fluorescens HK44 is the only species that approved to test in the field. It can degrade naphthalene with a mutagenized plasmid pUTK21 #Trögl2012.

Bioremediation with radioresistive bacteria
In 2000, Hassan Brim et al. developed a genetically modified bacteria to biodegrade the mercury ions Hg(II) in radioactive contaminated sites. The modified bacterium, Deinococcus radiodurans, is an extraordinary radioresistive organism that can survive under 12~16 kGy ionization radiation, while human can only survive less than 10 Gy. They cloned the mercury resistance gene MerA from E. coli strain BL308 into the pMD66 plasmid with four different insert designs. These four plasmids are capable to recombine themselves into the different places of chromosome to have different copy numbers of mer operon in cell. They showed that the mercury resistance can be regulated by the copy numbers of mercury resistance gene and existence of the promoter. The results also showed that the genetically modified D. radiodurans can biodegrade Hg (II) to Hg (0) under chronic gamma radiation #daly2000.

An engineered two-component system to sense lanthanide ions
The Salmonella PmrA/PmrB two-component system comprised PmrB, which is a cell membrane protein and has an iron (III) binding motif on the cell surface, and gene expression regulator PmrA, which can be trigger by PmrB. In 2013 Liang et al. replaced the iron-binding motif on PmrB with a known lanthanide-binding tag and made it to recognize lanthanide ions #Laing2013. Once the PmrB is activated by lanthanide ions, it would phosphorylate PmrA to induce the pmrC promoter. The modified two-component system was cloned into E. coli with two plasmids: pJBA25-pmrC and pBAD33-pmrA/pmrB. They showed that the activity of pmrC-gfp is promotional to the concentration of lanthanide ion. They also showed this system can be used to control cell mobility and could be a potential candidate for bioremediation #Laing2013.



Caffeinated coli for caffeine biodegradation
In 2013, Quandt et al. engineered E. coli and made it capable of biodegrading caffeine (1,3,7-trimethylxanthine). This strain can transform caffeine into xanthine, which is an alternative precursor of GTP or dGTP. They also broke the de novo guanine synthesis pathway to make E. coli addicted to caffeine. They first cloned the entire decaffeination operon from Pseudomonas putida CBB5 into E. coli plasmid pDCAF1, and knocked out guaB protein, which is crucial for de novo guanine synthesis. However, the cells were not able to survive with caffeine or theophylline. They refactored the plasmid by deleting the uncharacterized genes in the operon, inserting a stronger promoters and optimizing the DNA codon. This reconstructed plasmid pDCAF2 was able to survive with theophylline but not caffeine. They concluded that the operon was not able to remove the N7-methyl group. This activity was found to associate with the NdmC protein, which was co-purified with a glutathione S-transferase that encoded by orf8. A homologous coding region, gst9, was added to the end of the operon on pDCAF2 to construct pDCAF3. E. coli with pDCAF3 was capable to grow with either caffeine or theophylline. They also found the saturated cell density is correlated with the concentration of added caffeine, and this can be a sensor for measuring the caffeine level in different beverage #barrick2013.

iGEM project
The caffeinated coli is the iGEM project of UT Austin in 2012. This project was discussed in the above paragraph. Another iGEM project in 2012 designed a system that can indirectly detect plastic wastes in the ocean and trigger the expression of a polyethylene degradation protein (http://2012.igem.org/Team:University_College_London/BioBricks).