Pollution of the environment threatens life on this planet. Plastic waste has accumulated in our environment over the past 50 years, with about 67 million tons of plastic waste currently produced in Australia each year.*
Nanoplastic particles are not only routinely detected in the guts of aquatic animals but also in humans (including children) with unknown impacts of our health. Urgent action is needed to reduce the release of plastics into the environment. Clean-up campaigns and recycling have had limited success to date. The most promising strategy is to avoid petroleum-based plastic waste. However, modern society has become dependent on plastics.
A form of bioplastic known as polyhydroxybutyrate (PHB) offers a solution to this problem. PHB is used as an energy storage compound by some microbes, much like fat in mammals. This project is focused on selecting bacteria that have been isolated from agricultural environments (feeding of agricultural waste) and modifying them genetically so that they over-produce PHB. When these bacteria are grown in large fermentation tanks filled with agricultural waste, they can then be harvested and their PHB extracted. This PHB can then be processed to form an alternative plastic that has the same properties as petroleum-based plastic. The major advantage is that PHB-based plastic is degradable by natural microbes in the environment and therefore avoids the accumulation problems associated with petroleum-based plastic. Furthermore, PHB-based plastic can be used as a clean and 100% renewable energy source.
Another polymer produced by certain bacteria is curdlan. Like PHB, curdlan can also be used as an environmentally friendly, biodegradable form of bioplastic. The production of curdlan from agricultural waste is also a major focus of this project.
The modern demand for plastic should be met with biodegradable plastic (referred to here as bioplastic) instead of petroleum-based plastic. The major problem with bioplastic from bacteria is the low production rates coupled to the high cost of feeding the bacteria and extracting the bioplastic. My project solves two of these problems by using bacteria that are already naturally feeding on cheap agricultural waste products and modifying these to make the production rate of bioplastic much higher.
Currently there are several different bacteria that are known to produce high amounts of bioplastic. However, these have the disadvantage of requiring expensive food, such as sucrose or maltose. On the other hand, the bacteria that are suitable for growth on cheap food produce little or no bioplastic. For this reason, genetic manipulation of bacteria commonly found in agricultural waste is the most promising approach to produce high quality bioplastic at minimal cost.
Such an approach seems very logical. The major weakness could be that the majority of research never leaves the laboratory. This is because of the many unforeseen problems that are encountered at the level of scaling-up production. For example, the high cost of food for the bacteria could deter scaling up of production.
- Bioplastics will contribute to the global food crisis by competing for food sources. My approach will use agricultural waste and thus not compete for food.
- Bioplastics are not fully biodegradable. This criticism holds true for some bioplastics. Bioplastic derived from plant material, for example, usually requires temperatures over 50°C for degradation. Bioplastics from bacterial PHB show the highest degradation rates of all bioplastic.
- Bacterial bioplastics are too expensive. That is certainly true. Currently, they cost about five to ten times more than petroleum-based plastics. The two problems contributing to the high cost are low yield and the need for expensive, high-energy food sources for the bacteria. My project aims to overcome these problems by improving production rates in bacteria and reducing costs.
- Bioplastics encourages people to litter. This is indeed a serious problem, which can not be fixed by bioplastic. PHB and curdlan are not the only solutions to the problem of plastic. Rather one of a set of solutions. People need to see the importance of being responsible with waste.
- Bioplastic contaminates recycling processes. This is certainly true. Bioplastic has the unfortunate concequence of complicating plastic recycling. Currently there seems no way to avoid this problem. As with the litter problem, we humans need to improve our plastic waste management.
The novelty is to genetically modify bacteria to greatly increase the production rate. Bacteria are selected for this modification that can survive very well on low-cost agricultural waste as a food source. With these two innovations, the production rate will be increased and the production cost will be decreased.
Since the current cost of bioplastic is about five times that of petroleum-based plastic, the research goal is to bring the cost of producing bioplastic to a level comparable to that of petroleum-based plastic. There are several examples of how genetic manipulation has improved bioplastic production in some bacteria. Therefore, the goal is achievable. The big question here is how to manipulate the genes, since previous manipulations have had problems with genetic stability. With my many years of experience with genetic manipulation of bacteria, including an impending patent, I believe I am in a position to manipulate the genes appropriately. The patent specifically addresses the problem that for most bacteria, there is currently no way of modifying bacteria to control gene expression via conventional methods. The patent will protect a novel strategy that allows control of gene expression for any bacterium.
If this project succeeds, it will help alleviate a major environmental problem. Governments around the world are developing plans to deal with plastic waste. China now refuses to accept plastic waste from Europe. Kenya does not want plastic waste from the USA. In Australia, large parts of the industry have committed themselves to reducing plastic waste (Plastic Pact). In Germany, not only the citizens, but also the plastic production companies now have to pay a fee for disposal of plastic waste. The European Union is considering installing a tax on petroleum-based plastic production. The trend is clear, democratic countries are seeing the need for a dramatic change, and alternatives are in great demand.
The production of bioplastic would provide an environmentally friendly alternative to petroleum-based plastic. If the production costs and quality of both plastics were comparable, consumer concern for the environment would likely favor bioplastic, and the environment would be protected and preserved. It would make modern civilization more sustainable, help to preserve animal species, and provide perspectives for future generations.
The interest in bioplastic is rapidly growing. Already, various research groups worldwide are working on plastic producing bacteria. Some of these are working on improving plastic production via genetic engineering. Others are focused on isolating new strains from agricultural waste that are capable of producing plastic. However, the new strains are quite resistant to genetic engineering. Until now, no research group has successfully modified bacteria that have been isolated from agricultural waste to overproduce PHB.
An example of research aimed at isolating new strains of bacteria that can produce PHB was published by Getachew & Woldesenbet (Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low-cost agricultural waste material, BMC Research Notes, 2016).
Genetic engineering to overproduce PHB has been somewhat successful, see for example this paper by Nikel, Almeida, Melillo, Galvagno and Pettinari, (New Recombinant Escherichia coli Strain Tailored for the Production of Poly(3-Hydroxybutyrate) from Agro-industrial By-Products, Appl Environ Microbiol, 2006). It is important here to distinguish between cheap agricultural waste product such as wheat stubble and limited availability of by-products from expensive and energy-intensive processing such as whey and corn steep liquor. Hence, the plastic produced by this bacterium is too costly because of the by-products. Furthermore, this study highlighted some of the common pitfalls associated with genetic alterations, such as instability over extended fermentation times and low productivity. Improvements in the engineering quality of genetic alterations are greatly needed.
The biggest challenges for bioplastic production using genetically modified bacteria is genetic stability. Unwanted mutations can arise that free the bacterium from the burden of plastic production. Such ‘cheater’ mutants are able to grow faster and out-compete the producers, eventually dominating the fermentation culture. This requires more research on finding ways to establish genetic stability over extended fermentation times of weeks instead of days.
Also important here is the risk level associated with the genetic modifications. All bacteria that I use in the lab are rated at the very lowest risk level possible, i.e., risk level 1 or S1. Bacteria in this category have never shown any indication of danger to humans, animals or nature. Level 1 bacteria are found in nature without causing any harm to us. Genetic modifications of such bacteria to over-produce PHB or curdlan do not change the risk level, except perhaps to make the bacteria less able to survive in their natural habitats. Thus the genetic modification of such bacteria presents zero risk for human and animal health.
The strategy to find a cost-effective method to produce bioplastic involves three basic steps:
- Isolate and identify one or more bacteria commonly found in cheap agricultural waste products such as wheat stubble or apple waste for example.
- Genetically manipulate the bacterium or bacteria to overproduce bioplastic.
- Test production on a larger scale (fermentation involving several tons of agricultural waste).
Bacteria commonly isolated from agricultural waste are well known, and at least one of them shows great potential for bioplastic production in my lab. This can be genetically engineered to increase production by at least several fold. Currently, the bottleneck for this strategy is point 2. Specifically, the question is how to “tame” or “domesticate” a bacterium that has previously only been interested in survival. This wild bacterium should be turned into a bioplastic over-producer. In general, this approach is not new. Humans have been domesticating plants and animals for millennia, with enormous success. New technologies and scientific advances in genetics and biochemistry allow us to pursue these questions at the molecular level. In general, the work of “domestication” is to build genetic constructs that are robust and efficient, and to convert the tremendous energy of bacterial growth into bioplastic production.
I’ve had over 20 years of experience in molecular biology and genetics of bacteria. The major challenge for this project is the genetic modification of relatively uncharacterized bacteria. I have found a way to control gene expression in a wide variety of bacteria. This is especially useful for the genetic engineering of uncharacterized bacteria. Furthermore, an important advantage of my approach is that the genetic modifications are stable. My experience in this area means that I am in a good position to tackle the challenge of modifying bacteria to produce high levels of bioplastic.