Plastic from bacteria

I'm on holiday at the moment, so today's post is another section from my long essay last year, about the potential uses of biorefineries. It was written for a more scientific-based audience so might be a little harder to decipher than my usual posts.

Bioplastics

Bioplastics are polyesters that accumulate intracellularly in microorganisms in storage granules. They are usually built up from hydroxyl-acyl CoA derivatives through a range of different pathways in different microorganisms. As they are both biodegradable and biocompatible they have found numerous applications within medical and surgical fields, as well as having a greater environmental advantage over petroleum based plastics. The main disadvantages of bioplastics for commercial use are their high production and recovery costs.

The most widely produced bioplastics are poly(3-hydroxybutyrate) and poly(hydroxyalkanoic acid), referred to as PHB and PHA respectively. These both contain different β-oxidation intermediates as monomers, which are enzymatically polymerised through a condensation reaction. The structure of PHA is shown below (the 'n' indicates that the section show below is repeated multiple times):The first bioplastic to be described was PHB, found in Bacillus megaterium in 1926 by Lemoigne. It is stored in polymer form in granules within the cell.In order to decrease the recovery costs of the PHB granules, several attempts have been made to produce the secreted monomers, for polymerization outside the bacterial system. This has been achieved by expressing recombinant genes in E. coli

There are a large number of PHA polymers, ninety-one of which have been fully characterised. They are produced by both Gram negative and Gram positive bacteria via at least five different metabolic pathways. The main enzyme involved in polymer formation is PHA synthase (of the α/β hydrolase family), which polymerizes the monomers by connecting the coenzyme A thioesters of one monomer to the hydroxyl groups at positions 3, 4, 5 or 6 of the acyl moiety of the second monomer. There are four classes of PHA synthase, which are distinguished by their primary structures, substrate specificity and subunit composition. PHA synthases are found on the surface of the PHA storage granules, along with other proteins, and phospholipids.

(The structure of a PHA granule is shown above, image taken from Rehm 2003)

Engineering of recombinant bacteria that are capable of producing bioplastics requires both the transfer of a functional PHA synthase enzyme (there is no evidence as yet to suggest that any post-translational modifications of the enzyme are important for its function), and the engineering of suitable substrates that provide the enzyme with suitable substrates and sufficient concentrations. While the enzyme has been successfully transferred into model organisms such as E. coli, S. cerevisiae and even some transgenic plants, the provision of substrates is a more difficult problem as it involves dealing with large numbers of interlinked metabolic pathways. Metabolic flux analysis, carried out in transgenic E. coli, has substantially increased the carbon flux towards the production of PHB without detriment to the health of the bacteria, however this form of analysis has not yet been carried out on more complex PHA polymers.

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Madison LL, & Huisman GW (1999). Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiology and molecular biology reviews : MMBR, 63 (1), 21-53 PMID: 10066830

Steinbuchel, A., & Valentin, H. (1995). Diversity of bacterial polyhydroxyalkanoic acids FEMS Microbiology Letters, 128 (3), 219-228 DOI: 10.1111/j.1574-6968.1995.tb07528.x

Rehm BH (2003). Polyester synthases: natural catalysts for plastics. The Biochemical journal, 376 (Pt 1), 15-33 PMID: 12954080

Second Generation Biofuels

As part of my course last year, I wrote an extended long essay concerning the use of bacteria in biorefineries. As I've had a very lazy weekend (and to celebrate crossing the hundred post mark) I've decided to reproduce some of it here. More may be forthcoming at some point, depending on the laziness of my weekends.

Second Generation Biofuels

Second generation biofuels consist of lignocellulose material, which is broken down into simple sugars via enzymatic reactions and then fermented to produce ethanol. As lignocelluloses can be found in inedible plant matter (e.g corn husks, rice stems, and wheat stalks) they have the advantage that, unlike first generation biofuels, their utilisation does not compete with food production.

The three main components of lignocelluloses material are celluloses, hemicelluloses and lignin. These cannot be fermented directly and must therefore be broken down:The pre-treatment of the biomass is necessary both to remove lignin (although effective ligninases have been found in white-rot fungi, their rate of product turnover in bacteria is still too slow to be commercially successful) and to partially break down the cellulose to allow easier digestion by microbial processes. As the pre-treatment consists of harsh chemical processes, it would be advantageous within a biorefinery to use microbes which are able to withstand high temperatures and low pHs. For example, the cloning of thermostable cellulases into Trichoderma reesei allows a higher hydrolysis temperature compared to commercial Trichoderma enzyme, reducing the energy needed to cool the system after pretreatment with steam. The ability to save energy in this way could have a large economic impact, making the biorefinery more commercially feasible.

Currently one of the most popular microorganisms for use in lignocellulose biofuel production is Clostridium thermocellum which has an optimum temperature of around 60°C and also contains a cellulosome; a multi-protein cellulose-degrading complex attached to the bacterial cell wall. Cellulosomes are found in several bacteria, both Gram negative and positive, although they can differ in their structure and organisation (particularly of the cohesins and dockerins).
As potentially the entire process of ethanol production from lignocelluloses could be carried out by the microbes within a fermentor, the use of second generation biofuels in biorefineries has generated a lot of interest. The three main economic obstacles are the high processing costs, the narrow margin between biomass and fuel prices, and the large capital investment needed to initiate a cellulosic biorefinery. This could however, be overcome by increasing the potential for the production of high-value goods alongside the biofuel, either by adding pathways for the production of oleochemicals or bioplastics to the fermenting bacteria, or by utilising the lignin. This would provide the biorefinery with a greater capital return.

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Gilbert, H. (2007). Cellulosomes: microbial nanomachines that display plasticity in quaternary structure Molecular Microbiology, 63 (6), 1568-1576 DOI: 10.1111/j.1365-2958.2007.05640.x

Blumer-Schuette, S., Kataeva, I., Westpheling, J., Adams, M., & Kelly, R. (2008). Extremely thermophilic microorganisms for biomass conversion: status and prospects Current Opinion in Biotechnology, 19 (3), 210-217 DOI: 10.1016/j.copbio.2008.04.007

Zhang, Y. (2005). Cellulose utilization by Clostridium thermocellum: Bioenergetics and hydrolysis product assimilation Proceedings of the National Academy of Sciences, 102 (20), 7321-7325 DOI: 10.1073/pnas.0408734102

Biorefineries

Part of my course this year involves writing a Long Essay (3000 words, not really that long, but longer than most of the other ones we write). We had a choice of three, and luckily one was about bacteria, so I sort of jumped on it.

"To what extent could microbes be used as "biorefineries" that is, in biological processes for producing chemicals, fuels and polymers?"

I've been writing it for a while now, so I've come to know quite a bit about potential biorefineries and how they could work. The basic principle is that you get bacteria that you've engineered in various ways to produce a range of exciting chemicals, stick them in a fermentor, remove the chemicals at the end and sell them.

They're basically meant to work a bit like oil refineries, except using microbes and easily accessible raw materials rather than chemical processes and oil. Currently the most exciting idea at the moment is to use lignocellulose materials, that is all the bits of plants that don't get eaten (corn husks, rice stalks, bits of tree etc) in order to produce ethanol, to be used for fuel.

The closest thing that exists to biorefineries at the moment are the big ethanol refineries in the USA and Brazil. These produce ethanol from sugary substrates, sugar cane lees and maize at the moment. The major disadvantage of these is that they compete with available food stuffs, which is why there is presently the emphasis on using non-edible materials.

Lignocellulose contains two main chemicals; lignin and cellulose (biologists are not that imaginative sometimes). Lignin can't really be broken down by bacteria as yet, although there are some fungi that will do it. Cellulose can be, but needs specialist enzymes. To remove the lignin, and start breaking down the cellulose, some pretreatment is needed, mostly acids and steam.

The ideal microbe to use therefore, would be one that can break down cellulose and withstand the at least a certain degree of pretreatment, in order to reduce the cost of cooling and neutralising everything. The best one so far is Clostridium thermocellum which works best at 60 degrees C and has very efficient cellulose-digesting enzymes all held together in a complex, the cellulosome.

But when you look for current research on C. thermocellum, the very first thing that hits you is that, well, there isn't very much of it. In fact, there's hardly any of it, especially not heavy-duty 'what would this be like in a biorefinery situation' research. There's a couple of token papers about various aspects of it, and then just nothing. A spookily large amount of nothing.

There is, of course, a standard reason for this (and it doesn't involve conspiracy theories). The people who want to potentially build a biorefinery will want to make money out of it. They want to market the technology, or build a biorefinery with their own special bacteria, not tell every other competitor out there what they're doing.

From the point of view of an economist or a businessman it's a perfectly sensible thing to do. From the point of view of a researcher it's all slightly frustrating because it meant that there's a high probability that several people are all doing exactly the same experiments, and not telling each other. *sigh*