Thursday, January 31, 2013

Plastic bugs

Figure 1. Bacteria producing PHA. Art by Mark W. Slater
Here's where I thought we would get into some real biochemistry to explore the pathways involved in breaking down plastics. Turns out, the material gets a little heavy for the scope of this blog (lots of experimental conditions and little consensus) so there will be a little biochemistry and then we are going to turn to properly biodegradable plastics produced by micro-organisms. Lets first return to my favorite plastic, polyurethane and specifically the polyester type of polyurethane (PU) that is more amendable to bio-degradation (see the previous blog for help with understanding the why!).

It was evident from the beginning of polyurethane production and use, that the material was susceptible to fungal degradation. Tests were done with purified enzymes and in soil where fungal communities were discovered to have a number of PU degrading species. However the best results were obtained when a biostimulant was added such as Impranil DNT, a readily digestible form of  PU - sort of like baby formula for fungi. The addition of cultures of known PU biodegrading fungi was also helpful in increasing biodegradation of PU by native soil fungi. The enzymes were thought to be extracellular (i.e. pumped out of the fungus) and to be a combination of ones that could cut bonds in the middle of the polymer at random positions (endoenzymes) and those that chopped off monomers from the end of the polymer chain (exoenzymes). In general, an additive like yeast extract also needed to be added as a carbon source to create  more extensive degradation, though fungi that could utilize PU as the sole carbon source were identified (Howard et al., 2012) Recently, an endophytic fungus from the Amazon rainforest has been identified that is able to efficiently use PU alone as a carbon source without additives (Russell et al., 2011). Although this is promising, it has as only been tested in small scale laboratory experiments (with Impranil DNT) and I'm not sure how it compares with the fungi that were identified earlier (Howard, 2012).

On the bacterial side, several  species have been identified as degrading PU. Enzymes from the Pseudomonas genus have been characterized and fall into a class that resemble lipolases which are enzymes that degrade lipids (fats). Interestingly, when the sequences of the genes are compared to each other and to other lipolases, it was shown that they come from genetically diverse backgrounds and do not have a common parent. This suggests that several branches of the evolutionary tree thought up the same idea at different time points (Howard, 2012). 

However, all these test have been done under laboratory like conditions on a small scale and the general impression from the literature is that most plastic that is currently produced cannot be degraded sufficiently efficiently to make industrial efforts economical. Efforts are therefore turning towards more ecofriendly biodegradable plastics and also to plastics produced from renewable sources (biopolymers) rather than from petrochemicals. So lets leave the degradation of plastics and turn to plastic biosynthesis. 

Figure 2. PHA granules in bacterium (Kunasundari and Sudesh, 2011)
Since we are in the business of investigating what micro-organisms can do for us, is it really true that bacteria or fungi can make plastic? YES IT IS!  Many biopolymers  use micro-organisms in a fermentation step to breakdown a feedstock such as corn or mollases to produce a monomer. Examples of these plastic monomers include lactic acid used in polylactic acid (PLA) plastic and succinic acid used in Poly(butylene succinate adipate)( PBSA) (Wolf 2005). However, only one type of plastic [polyhydroxy alkanoates (PHA)] is produced directly by bacteria (Figure 1 and 2). Just like there any many types of PU or polyethylene, there are many types of PHA. Monomer units of different types of PHA are shown in figure 3. Polyhydroxybutyrate (PHB/PH3B) which is a PHA, was first discovered as far back as 1926 (Castilho et al, 2009), but because of the expense of production and the lack of technology and the bliss of  ignorance of the consequences of using oil for everything, the discovery languished. Recently with making our world into a rubbish pit by filling it with landfills, the finite supply of oil and the worsening consequences of churning out CO2, much more effort has been devoted to research into bringing down the costs of bioplastic production. 
Figure 3. Monomer units of different PHAs
As we are environmentally focused (reduce, reuse, recycle) lets first look at the use of inexpensive feedstocks as one of the aspects for bringing down production costs of PHA.  Feedstock can either be liquid and used in submerged fermenation processes (SMF) or from more solid material used in solid state fermentation (SSF) processes. SMF is where the liquid feedstock is mixed in with the bacteria in one large agitating container. A few SMF feedstocks include molasses and sugar cane liquor (by-products of sugar production), sugar (which while not as cheap as molasses is less expensive than glucose), starch based substances such as wheat pearlings (the bran husk from the wheat kernel that is removed by rollers in a process known as pearling), cellulosic materials (e.g. sugar cane bagasse hydrolysate; the leftovers from sugar cane stalks after the sugar has been removed), hemicellulosic substances (e.g. xylose; a monosaccharide in hemicellulose found for example in corn husks and wood pulping byproducts) and whey based waste material (a large and mostly wasted resource from the dairy industry). SMF feedstocks also include organic waste matter such as swine waste liquor, malt waste from spent barley, millet refuse from brewing brewing and waste from olive oil mills. There is therefore great potential to couple some of these industries with PHA production. SSF feestocks on the other hand, used moist solid particles in substrate beds where the feedstock is the support surface (i.e. the surface on which the bacteria grow). SSF is a potential alternative for disposal of agricultural waste such as rice bran, cassava bagasse and cakes from vegetable oil extraction. Instead of needing to dispose of these via incineration or landfill, they could become an extra source of revenue.

PHA productivity in grams of PHA per kilogram feedstock per hour can range from 0.02 to 2.57 g/kg/h (Castilho et al, 2009). The top producer in the article by Castilho et al., 2009, used recombinant E. coli containing PHB genes from the bacterium A. latus in a whey + salts + citric acid + trace metals SMF fed-batch reactor (read on for what this is!). The experiments were done by Ahn et al, 2000. A recombinant micro-organism means that it has an engineered genetic composition compared with its original state. In this case, a plasmid with PHB synthesis genes from the bacterium Alcaligenes latus (now known as Azahydromonas lata) were added to the E. coli bacteria on a plasmid. A plasmid is a (relatively) small piece of circular DNA that contains all the necessary components for autonomous replication within the bacteria. Genes of choice can be inserted into the DNA of the plasmid and the plasmid can then be inserted into a new bacterium so that the effect of the genes can be studied. Certain strains of E. coli are laboratory work horses, allowing the genes of more obscure, more finicky bacteria to be studied more easily. The autonomous replication property of a plasmid means that several copies of the plasmid exist within the bacteria and when the bacteria divides, some of them will remain in each daughter cell, ensuring that the bacteria retain the ability to synthesize PHB.

So now we have the E. coli PHB factory, what happens next?  Ahn et al, 2000 use a fed batch reactor. This is a container with a stirring mechanism and probes by which the oxygen and pH can be monitored. At the beginning of fermentation, less than half the maximum volume of culture was added. As the lactose concentration fell (lactose is the sugar carbon source utilized from the whey by the bacteria) the pH rose and more whey feedstock was added to bring the pH back down, thus increasing the volume in the reactor. The authors found that the oxygen concentration had a significant role in PHB productivity. The growth of the bacteria could be divided into 2 phases; a fast growth phase where high (40%) oxygen was optimal to achieve high bacterial density and a slow growth phase with lower oxygen levels where PHB production increased. This makes sense as PHB is stored as an energy reserve during lean times like low oxygen levels. They also found that PHB production was highest during oxygen lowering so that with a step wise decrease in oxygen concentration from 40% to 30% to 15%, the highest PHB productivity of 2.57g/kg/L was achieved. The only disappointment for me with this study, was that the whey feedstock was not obtained from a dairy but from bought whey powder so the experiments was  not as "real" world as the could have been. 

After expression in bacteria, the PHA must be extracted and this is estimated to be up to 50% of the cost of PHA production (Fiorese et al, 2009). In comparison with biosynthesis, the extraction process has been much less researched. Kunasundari and Sudesh, 2011 provide a review. The extraction process consists of pretreatment of the collected bacteria to weaken the cell wall and cell membrane (for an understanding of bacterial cell structure, take a look here). Following pretreatment, an extraction process is used to isolate the PHA from the other cell components. Extraction has commonly been done with organic solvents such as chloroform and this clearly presents enviromental consequences as well as being uneconomical unless the solvents can be recycled. Fiorese et al, 2009 use 1,2-propylene carbonate in the extraction process which is a much less toxic solvent than something like chloroform and can also be recycled, thus reducing costs. Major factors of any extraction process are purity, the affect on the homogeneity and breakdown of the PHA (i.e. shortening of the plastic polymer molecules). The standards for these qualities will depend on the end product for which the PHA is to be used. 

Other extraction procedures being investigated include chemical or enzymatic disruption and mechanical disruption. Each method has advantages and drawbacks. For example, enzymatic digestion involves mild conditions and therefore provide an environment for a high molecular weight, high homogeneity and pure end product. On the downside, enzymes are expensive to produce and have finite life times. Mechanical disruption is advantageous because no chemicals are involved, therefore no contaminants are added that need to be removed later and there are no added polluting solvents to deal with. However, there is a high capital investment cost of the mechanical disruption machinery, processing times are long to ensure maximum disruption and scale up is difficult. As of 2009, PHA plastic is between 3-17 times more expensive to produce than petrochemical based plastics (Castilho et al., 2009).

Having said that, there are a couple of companies producing PHA industrially. Metabolix in Cambridge, MA, USA, produced 50,000 tons of its PHB product Mirel in alliance with ADM between 2004 and 2012 (it has since closed). "Meredian, Inc has a pilot plant in Georgia, USA that produces 13,600 metric tons per year of PHA. Construction is beginning at the site for a plant that will produce 91,000 metric tons per year of PHA. Tianjin Green Bioscience and DSM operate a 10,000 metric ton per year PHA plant in China. Tianan Biologic Material Co. is boosting its capacity in China to 10,000 metric tons per year. About a dozen other companies operate lab or pilot scale PHA facilities"(Smock, 2012).

Can PHA plastics be used in the same way as petroleum based plastics? Currently, the main application of PHA is used for making thin plastic films such as plastic bags and food packaging which is an application that otherwises uses polyethylene or polyurethane plastics. With further research, PHA plastics could also be used for other products such as tissue engineering materials, bioimplant materials and smart materials (Chen, 2010)The major advantage of PHAs produced from biowaste, especially when coupled with a dairy or sugar production plant for example, is that they are easily biodegradable. When (yes I do say when) the cost of their production is brought down sufficiently and their properties altered, they could be a significant source of plastic. 

While this post has been about PHA (mainly), biopolymers produced from waste products initially broken down by micro-organisms and then made into polymers through other processes, is also an alternative technology. Some of these biopolymers are not biodegradable. In 2005, the European Commission provided an interesting (and long) report on the "Techno-economic Feasibility of Large scale Production of Bio-based Polymersin Europe" (Wolf, 2005). It provides exactly what the title says and generally comes out on the side of biobased plastics as necessary for the environment, company reputations (being green is good marketing) and for scientific innovation, highlighting that green technology is about much more the reducing our environmental impact and the safety of future generations - as if this wasn't enough! 

Next post: Feb 28th. Right now the subject is a toss up - more on PHA or heading to the ocean to look at our plastic pollution there. 


Ahn, W., S. Park and S. Lee (2000). "Production of poly(3-hydroxybutyrate) by fedbatchculture of recombinant Escherichia coli with a highly concentrated wheysolution." Appl. Environ. Microbiol.. 66: 3624-3627.

Castilho, L. R., D. A. Mitchell and D. M. Freire (2009). "Production of polyhydroxyalkanoates (PHAs) from waste materials and by-products by submerged and solid-state fermentation." Bioresour Technol 100(23): 5996-6009.

Chen, G.-Q., Ed. (2010). Plastics from Bacteria:Natural Functions and Applications (Google eBook). Microbiology Monographs.

Fiorese, M., F. Freitas, J. Pais, G. de Araga˜o and M. Reis (2009). "Recovery of polyhydroxybutyrate (PHB) from Cupriavidus necator biomass by solvent extraction with 1,2-propylene carbonate." Eng. Life Sci. 9(6): 454-461.

Kunasundari, B. and K. Sudesh (2011). "Isolation and recovery of microbial polyhydroxyalkanoates." eXPRESS Polymer Letters 5(7): 620-634.

Wolf, O., Ed. (2005). Techno-economic Feasibility of Large scale Production of Bio-based Polymers in Europe.

Lifestyle Magic: Unblocking your sink
The other day, I was confronted by a non-draining bathroom sink. I googled it up and read that spooning down a bit of baking soda followed by apple cider vinegar, does wonders. And it does. You don't even need a spoon. I poured baking soda down, followed by the vinegar and enjoyed the frothing reaction. After several pours, the sink drained beautifully, not an ugly chemical in sight.

Tip about baking soda shampoo from last post. I found that my hair does best on 1.5 tablespoons of backing soda to 2 cups of water. Less or more gives a less glossy result. Your hair could be different! 

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