Biodegradation of carbon nanotubes

Dr. Neil Canter, Contributing Editor | TLT Tech Beat April 2009

Researchers have discovered a naturally occurring enzyme as a potential candidate that can degrade these unique nanomaterials.

KEY CONCEPTS
• A naturally occurring enzyme known as horseradish peroxidase has been found to biodegrade carbon nanotubes.
• Used with hydrogen peroxide, HRP degrades carbon nanotubes over a 16-week period at 4 C. The process accelerates at elevated temperatures.
• The active component doing the decomposition is an iron (IV) porphyrin radical cation formed from the oxidation of HRP by hydrogen peroxide.

The strong interest in nanotechnology and the development of new nanomaterials has led to some concern about their potential toxicity. Nanomaterials are typically between 1 and 100 nanometers in size. As a comparison, the diameter of one human hair is 10,000 times larger than 1 nanometer.

As more and more commercial products have been developed with nanomaterials, concern has arisen about their safe use, particularly when placed in contact with human beings. For example, titanium dioxide is used widely as a pigment in cosmetic products such as sunscreens. Conventional titanium dioxide is white in appearance while nano-sized titanium dioxide is transparent. Does conventional titanium dioxide used in the past have a comparable health and safety profile to titanium dioxide nanoparticles?

One notable nanomaterial is carbon nanotubes, which is composed of thick rolls of graphite that are also stronger than steel. In a previous TLT article, the precursor to carbon nanotubes, graphene, was found to be the strongest material ever examined (1).

Carbon nanotubes have found use in a variety of applications, including reinforcing plastics, conducting electricity in electronics and as sensitive chemical sensors. Alexander Star, assistant professor in the department of chemistry in the University of Pittsburgh’s School of Arts and Sciences, says, “The toxicity of carbon nanotubes is controversial. There are some reports that carbon nanotubes, if inhaled, can cause respiratory inflammation in as similar manner to asbestos.” Other potential negative effects include formation of free radicals and peroxidative products.

But identification of a potential safety problem is more complicated because carbon nanotubes have been produced in many different forms. Star adds, “Carbon nanotubes have been found with different lengths and have been prepared in large bundles or ropes. Functionalization has also produced different nanotubes with new characteristics.”

With the growing use of carbon nanotubes, there is an increasing chance of environmental pollution and exposure. Development of a safe method for decomposing carbon nanotubes is desired. Such a technique has not been available until now.

NATURAL HORSERADISH PEROXIDASE
Safe decomposition of carbon nanotubes is quite challenging because of the nature of the material. Star says, “The carbon-carbon bonds in carbon nanotubes are the strongest found in nature and are extremely robust.”

The researchers identified the natural enzyme horseradish peroxidase (HRP) as a potential candidate to degrade carbon nanotubes. Star says, “HRP became a plausible option because it is known to biodegrade different organic compounds, including polyaromatic hydrocarbons that are close in composition to carbon nanotubes. This enzyme is also very robust and works well over a broad range of temperatures.”

The carbon nanotubes evaluated by the researchers were initially oxidized with a combination of sulfuric acid and hydrogen peroxide to remove residual metal catalyst. This step also carboxylated the carbon nanotubes to enable them to be soluble in an aqueous environment.

HRP was then added to the carboxylated carbon nanotubes in the presence of hydrogen peroxide and the mixture incubated for 16 weeks at 4 C at a pH of 7. Star says, “We used a low concentration of hydrogen peroxide because of its compatibility with HRP. The mixture was kept in the dark to avoid enzyme degradation and the photolysis of hydrogen peroxide.”

A number of different analytical techniques were conducted to evaluate the condition of HRP and determine the concentration of carbon nanotubes present in the mixture during the study. Electron spin-resonance analysis showed that the HRP remained viable and was not deactivated by the carbon nanotubes.

Over the 16-week period, aliquots of the mixture were removed for evaluation and were replaced by an equal volume of hydrogen peroxide. Star says, “Using the naked eye, we could see the concentration of nanotubes decrease from the beginning of the study through week 16. The initial gray color declined in intensity during that time and the mixture displayed more and more transmittance.”

Figure 3 shows samples taken at the beginning of the study and after weeks 1, 4, 12 and 16. Clearly the mixture is becoming more transparent over the duration of the experiment. Light scattering measurements verified the increase in transmittance. Degradation of the carbon nanotubes was confirmed by transmission electron spectroscopy, which showed a decrease in the length of the carbon nanotubes and the concomitant appearance of globular material.


Figure 3. The enzyme horseradish peroxidase (HRP) slowly degrades carbon nanotubes at 4 C over a 16-week period. Samples taken at weeks 1, 4, 12 and 16 become more transparent as the study progresses, which indicate the carbon nanotubes are being degraded. (Courtesy of the University of Pittsburgh)

The process can be accelerated by using higher temperature and larger concentrations of hydrogen peroxide. Star says, “Carboxylated carbon nanotubes were incubated with HRP at 37 C for five days with hourly additions of hydrogen peroxide. Over that time frame, 40% of the carbon nanotubes were decomposed. We believe this moves the decomposition of carbon nanotubes from weeks to days, but there is still room for optimization.”

The degradation has been attributed to the formation of a highly reactive intermediate. Star explains, “The active site of the HRP is an iron (III) porphyrin ring complex. Oxidation with hydrogen peroxide generates an iron (IV) porphyrin radical cation that decomposes carbon nanotubes by reducing their length.”

Work is in progress to better understand what species are formed during degradation of the carbon nanotubes. Star says, “We are using mass spectroscopy to identify the molecules present and will use HPLC to isolate the individual components.”

Future work will involve using HRP on other carbon nanotubes that have different lengths and different structures such as number of walls. Details about the research can be found in a recent article (2).

The work described here is an important step in dealing with potential concerns that will arise from the increasing presence of nanomaterials in consumer and industrial products.

REFERENCES
1. Canter, N. (2009), “Graphene: The Strongest Material Ever Examined,” TLT, 65 (2), pp. 28–29.
2. Allen, B., Kichambare, P., Gou, P., Vlasova, I., Kapralov, A., Konduru, N., Kagan, V. and Star, A. (2008), “Biodegradation of Single-Walled Carbon Nanotubes through Enzymatic Catalysis,” Nano Letters 8 (11), pp. 3899–3903.

Neil Canter heads his own consulting company, Chemical Solutions, in Willow Grove, Pa. Ideas for Tech Beat items can be sent to him at neilcanter@comcast.net.