Saturday, August 24, 2013

Fuel for Biofuel 3: Cellulosic feedstocks

In the last post, we established that cellulosic feedstock are somewhat more complicated in their molecular structure than a corn feedstock.  What are the main feedstocks being considered for commercialisation? What are the fuels they are used to produce?

Corn and sugarcane are known as the feedstocks for first generation biofuels. Other feedstocks are called advanced biofuels but these still consist of ethanol and biodiesel. Biodiesel is produced from the triacylglycerides obtained from soy, oily seeds or the mesocarp of palm fruits (click here for illustration). More technically, a process known as trans-esterification converts the triacylglycerides to fatty acid methyl esters that can be directly used in engines. I hope to post on this too!

So what are the feedstocks current being exploited for advanced biofuels?

C3 vs C4 vs CAM plants
First, lets have little more plant education. One way in which plants can be classified is according to differences in their photosynthetic pathways. Photosynthesis is the use of the energy from sunlight to convert CO2 to an organic carbon molecule together with the release of oxygen.  With this type of classification, 3 groups occur: C3, C4 and CAM. The first molecule produced by the use of CO2 in C3 plants is a 3 carbon molecule and a 4 carbon molecule in C4 plants. C3 plants operate more efficiently under cool wet conditions while C4 plants are more water efficient and well adapted to dryer and hotter conditions. CAM (or Crasseulean Acid Metabolism) are even more water efficient and survive well in arid dessert climates. One of the ways in which water loss is minimised is through regulation of the gas exchange pores on the underside of the leave; these are called stomata. In C3 plants they are open all the time, in C4 plants stomata open only during the day when photosynthesis can occur but in CAM plants, stomata open only during the cooler night. CO2 is gathered and stored as crasseulic acid during the night and then used during the day. As water loss can occur through the stomata, regulating when they are open, regulates the loss of water. Most plants are C3 ( rice, wheat, soybeans, potatoes, beans, fruit trees). C4 plants include corn and many annual summer plants. CAM plants include catuses and agave plants.This is of course simply put. More details can be found in this article.

Non-woody perennials.
Popular contenders for cellulosic feedstock plants are the perennial grasses such as miscanthus, switch grass and elephant grass are all C4 plants and are more efficient at photosynthesis than C3 plants.

Additionally, their root system consist of a network of rhizomes that store energy and nutrients for the following season of growth allowing a fast regeneration time, drastically decreasing the amount of fertilizer needed and preventing soil erosion.  Further the perennial grasses under consideration are native to Amercia (including switch grass, Praries grass and Big and Little Bluegrass, suggesting that the Northern Plains and Southeastern grasslands could be a source of advanced biofuel.

Woody perennials. 
Woody perennials include fast growing trees such as Willow, SweetGum and Cottonwood.
Sweet Gum

Like the perennial grasses they have several advantages over energy crops such as corn. First the amount of biomass produced for the same land area is much greater, they have better water use, don't require fertilization and deep root systems maintain soil structure and prevent erosion.  Secondly, some species such as willow and eucalyptus are amendable to the technique of coppicing, where the plants are cut down to near ground level every 3-5 years. This allows for increased biomass and these species rapidly regenerate from the storage system contained in their roots.

New bioenergy crops.
With modern agricultural practices and deforestation, large tracts of land have become semi arid. Plants such as the agave that uses CAM photosynthesis, is able to grow in such conditions and could be used to regenerate these semi-arid areas while providing a biofuel feedstock. Agave plants of different species have been under cultivation for many 100's of year for alcholic beverages (think Tequila!) and for sisal fibers to use in twine, paper, dartboards (!), handicrafts and mattresses for example. Sisal fibers could be a potential source for a biofuel feedstock.

Another problem with modern agriculture is the salinization of irrigated land and it is estimated 1-2% of irrigated land is lost every year. Highly salt tolerant plants such as prairie cordgrass and Eucalyptus species are potential feedstocks and could also be used to desalinate lost arable land.

Waste matter 
When I started considering biofuels from lignocellulosic feedstock, I was hopeful that a large proportion would be generated from the waste products of current industrial processes. To some extent this is true.
Firstly there is corn stover. Stover are the leftover inedible fibrous stalks and leaves after the cobs have been harvested. It is estimated that 13.5 billions gallons of ethanol could be generated from this stover. And while the bagasse produced after sugarcane harvesting in Brazil is not used as a feedstock but is burnt and leads to production of 2giga watts net electricity, this can also be classed as biofuel, just like wood burnt in your fireplace. However the costs of collection and transportation to processing plants of corn stover and the loss of nutrients and risk of soil erosion could make this economically and environmentally untenable though this view depends on where you get the information.

Many other crops such as wheat, rice and soybean leave a waste product called residue after the harvest. This is also a potential source of biofuel feedstock but is complicated by the same issues that are involved in corn stover collection in that the decay of the residue returns nutrients to the soil and prevents soil erosion by wind and rain. The ins and outs leading to an assessment of commerical viability are beyond the scope  of this post as they are many and complex. The 2011 US One Billion Update provides an in depth analysis of all the different aspects that need to be considered.

Wood waste
There are several sources of wood waste. The first if from timberland (forests used for logging) and this includes tree tops, branches, dead or rotten wood, small trees and non-commercial trees. This waste is normal left in the forest to decay but could provide a biofuel feedstock source. Careful management would be necessary as some of this waste wood provides a return of nutrients to the soil, habitats for insects and birds, fertiliser for new saplings and prevention of soil erosion.

Other wastewood comes from forest thinning in timberland areas and non-logging forests where biomass is removed to reduce the risk of forest fires becoming catastrophic. Waste wood is also generated when timberland is converted to non-forest land such as cropland and pasture roads. A last source of waste wood is from urban wood which includes furniture, landscaping, remodeling and construction. Much of this ends up in landfills. To me, that seems like a wonderful source for generating biofuel.

In the 2011 US One Billion Update, it is estimated that wood sources will produce up to 244 million tons of dry biomass.

Municipal waste (MSW)
This is the matter that everyone discards on a daily basis. Food scraps, paper waste, plastic etc.  From the point of view of biofuels, the most valuable items are paper and food. These need to be separated from the rest so despite the presence of a collection and transport system for MSW, the processing necessary in order to use it commercially as biofuel feedstock, is complicated. 

Animal Fat and Yellow grease.
Animal fats are generated from the slaughter of animals. However they are less valuable as a feedstock as they have a tendency to crystallize at lower temperatures. Yellow grease is the oil left over after cooking for example in restaurants and is a mixture of plant and animal fats. It is in limited supply but could be commercially viable for smaller biofuel operations.

The table shows the total and projected tons of biomass available from different feedstocks for ethanol and biodiesel (from 2011 US One Billion Update)

Current commercial operations
According to Environmental Leader, there are 11 competitive cellulosic ethanol plants currently operating and use of cellulosic feedstocks will be cost competitive with corn as a feedstock by 2016 and in fact many corn based plants are shutting down due to eroding profit margins. Wikipedia provides a table showing several companies in the US, their start dates and production capacities. A selection is shown in the table below and the feedstocks show that corn stover, wheat straw, wood waste and municiple waste are among the main ones in use.

Company Location Feedstock Capacity (million gal/year) Began Production Type
Abengoa Bioenergy Hugoton, KS Wheat straw 25 - 30 [33][34] est. late 2013 Commercial
DuPont Nevada, IA Corn stover 30[41] est. 2014 Commercial
Fulcrum BioEnergy Reno, NV Municipal solid waste 10 est. end of 2013 Commercial
Gulf Coast Energy Livingston, AL Wood waste 0.3 [42] before 2008 Demonstration
Mascoma Kinross, MI Wood waste 20 est. 2014 Commercial
POET LLC[43][44] Emmetsburg, IA Corn stover 20 - 25 est. lat 2013[45] Commercial
POET LLC[46] Scotland, SD Corn stover 0.03 2008 Pilot

Concluding...this post
Cellulosic feedstocks are on a roll and there are many sources. I hope to have a closer look at these in coming posts and to bring in the bugs!

References and websites used:
Youngs and Somverville  2012, Development of feedstocks for cellulosic biofuels. F1000 Reports Biology. Volume 4, Issue 10.
Environmental Leader
Bloomberg New Energy Finance
Biofuels Digest
US One Billion Update
Wikipedia; various

Tuesday, July 9, 2013

Fuel for Biofuels part 2: cellulosic feedstock

In Fuel for Biofuels part 1, I took a look at producing biofuel from corn. This has come to be known as first generation biofuel. The next generation biofuels can be produced from cellulosic feedstocks and by aquatic algae. In this part 2, I'm going to take a look a cellulosic feedstocks. In an attempt to make the posts more digestible, I'm going to make them shorter and hopefully more frequent.

Previously we learnt that corn is a commercially popular feed stock because it is easily broken down having a relatively simple structure and requiring simpler chemical and enzymatic degradation processes. Cellulosic ethanol feedstocks include sawdust, forest thinnings, waste paper, grasses, farm waste (e.g., corn stalks, wheat straw, and rice straw), switchgrass and other perennial grasses. Where is the cellulosic material found in plants?

Plant cell wall macro structures
There are many differences when plant cells are compared with animal cells, such as the presence of chloroplasts and a large central vacuole but the difference we are most interested in, is the cell wall which animal cells lack. The cell wall is one of the features of plants that gives a plant rigidity.

The food generating cells of leaves have a thin cell well and these cells are known as parenchyma. The cells that make up young flexible stems are known as collenchyma. Their cell walls are thicker particularly at the corners where several cells intersect. The thicker the cell wall the less nutrients can get into the food production centers but collenchyma cells despite their thicker cell walls, are still alive. The cell wall consists of cellulose,  hemicellulose and pectin. Collenchyma cells are able to stretch as the plant grows. Figure 1 illustrates the plant cell wall structure. You can see that the cellulose is arranged in microfibrils and a magnified illustration of a microfibril is shown in figure 2. The highly regular formation of the fibrils can easily be imagined (curiously it reminds me of the highly structured formation of skeletal muscle).  Hemicellulose links the microfibrils together. In figure 1 cell wall illustration, you can also see a molecule called pectin. Pectin is another polysaccharide  and works to both give mechanical strength to the cell wall and to improve impermeability.
Figure 1 Structure of plant cell wall (Link)

Figure 2 Structure of cellulose microfibril (link).

Figure 3 Electron micrograph of cell wall with secondary cell wall (Link)
To the inside of the primary cell wall in these cells, a secondary cell wall can be laid down and toughened by the deposition of lignin. Lignin is water impermeable and is very rigid, with great mechanical strength; without water the cell cannot survive and dies. This layer of cells becomes known as sclerenchyma.

Figure 3 shows an electron micrograph of the cell wall and you can really see how thick the secondary wall is

Molecular structures 
We are interested in the structure of these polymers because they will affect downstream processing of the cellulosic materials for biofuel.

Cellulose consists of glucose monomers linked together by C1 and C4 as shown in Figure 4.
Figure 4 Monomer of cellulose
The number of glucose units can be in the 10,000s and its regular structure allows it to pack tightly together giving strength.

Hemicellulose on the other hand is amorphous and flexible with little structure strength. Hemicellulose consists mainly of 5 carbon sugar monomers such as xylose, mannose, galactose, rhammnose and arabinose. Figure 5 shows a picture of a common hemicellulose repeating unit with the different pentose sugars.
Figure 5. Comon hemicellulose molecular motif
Pectin, like hemicellulose, also consists of several different monosaccharides and illustration of pectin is shwon in figure with the different regions shown in blue and the monosaccarhides illustrated as small differently coloured polygons, and the key to the polygons is given in the figure.

Lignin is unusual in a structural polymer in being very irregular. It consist of three molecules called monolignol monomers: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol.  Different plants have lignin with different proportions of each molecule. Figure 6 shows an example of a possible lignin structure. It makes me think of lace.
Figure 6. Example of possible lignin structure.
So, wow, thats a bit more complex than the starch nolecule of corn. Interesting, the difference between starch and cellulose, both of which consist of chains of glucose molecules is the way in which the monomers are linked. In starch, animals have enzymes that can easily digest the link while they do not have the enzymes to easily digest the cellulose chemical links. How do we turn the complex structure of plant cell walls into biofuels? See the next post!

    Sunday, May 19, 2013

    Fuel for Biofuel part 1

    Figure 1: Illustration of bacterial ethanol (EtOH) production from corn by Natural Line Studio
    Last month we took a look at ocean plastic. It was pretty depressing and from the point of view of microbiology,  a bit of a non-starter.  So lets go back to land and biofuels, one of the next great hopes that makes use of various micro-organisms.
    Summarising the basics of the biofuel process
    In order for micro-organism to produce biofuel (Figure 1) they need a food source or feedstock. Feedstocks are generally made of starch (Figure 2) or lignocellulose (fibrous parts of plants). These are polysaccharides of small subunits like monosaccharides or disaccharides, just like plastics are polymers of hydrocarbon monomers (see my January post). In Figure 2 each hexagon is a glucose (monosaccharide) molecule. A row of glucose molecule joined together, by glycosidic bonds, in this way is called amylose. When the glucose from the top row forms a bond with a glucose in the second row, it is called amylopectin. Therefore starch consists of amylose and amylopectin.
    Figure 2. Structure of starch
    The first step is to break up the polymers so that the maximum number of chemical bonds are exposed and available to micro-organisms.  Mechanical, heat and enzymatic action is used to achieved the breakdown of the polymers to edible chunks (monosaccharides and dissaccharides). These breakdown steps are followed by and often coincident with the fermentation step where micro-organisms use the released sugars as a carbon source to produce ethanol.You can imagine that the cost of this is not insignificant, so making it as efficient as possible is key. Biofuels include ethanol, butanol, biodiesel and a number of second generation fuels. Currently ethanol is the only commercially produced biofuel, a so-called first generation biofuel and we will start with this.

    Ethanol is the main biofuel produced globally (Fisher et al,. 2008). Ethanol is an alcohol. Chemically, alcohols are denoted by a hydroxyl or -OH group (see structure in figure 3)
    Figure 3: Ethanol
    and it is added to petrochemical vehicle fuel to increase the octane number (see bottom of post for octane number explanation) and to cut down on smog inducing emissions. Flexible fuel vehicles have modified engines that are able to use mixed gasoline/ethanol as fuel. A common flex fuel is E85 (85% gasoline, 15% ethanol).  Sugar cane in Brazil and corn in the USA are the main feedstocks of bioethanol with very high conversion efficiencies (i.e. % of feedstock input converted to ethanol) of between 90-95%  (Fisher et al, 2008). The problems with ethanol is that it is rather different from gasoline. But hold on....just what IS gasoline or petrol? It consists of a mix of hydrocarbons called alkanes, cycloalkanes and olefins with 4-12 carbons in each molecule (Figure 4). Additives are also present to enhance the use of the fuel in an engine such as MBTE in Figure 4. Structurally and chemically, these molecules are quite different from ethanol.
    Figure 4: Common components of gasoline

    Since most cars can run on 10% ethanol mixed with gasoline, why does flexfuel only make up only 6% of fuel used (Solomon, 2010)?. The infrastructure present for piping gasoline around the country is extensive and obviously has taken considerable investment. Because ethanol can be corrosive to the materials of this infrastructure as well as to unadapted car engines (Fisher et al, 2008), a new infrastructure would be required specifically for flex fuel and adapting cars costs a couple of thousand dollars, something many people can't afford.

    The other big problem is the use of corn as a feedstock despite its good conversion efficiencies. This is because, when considering the impact of feedstocks for biofuel, there are numerous factors to consider apart from the hopeful reduction in climate change gases. These include water use, land use prior to corn growth, biodiversity decrease, pollution of water and air due to growth (pesticides, fertilisers), processing (needs electricity from oil) and socio-economic impact. There have been numerous studies on corn as an ethanol feedstock with varying outcomes. The overall message that comes through is that although corn as a feedstock is better than using petrochemcials, it is unsustainable and even at maximum capacity, could only support 20-30% of biofuel needs of the US (Solomon, 2010). Though sugar cane as a feedstock fares better than corn, there are other feedstocks known as cellulosic (sometimes called lignocellulosic) material (includes paper, wood, cardboard, fibrous plant material) that have a much lower impact as they can come from waste products of current industries, not from the new growth of a dedicated crop. Interestingly, one of the main public concerns of corn as an ethanol feedstock is the increase in food prices as more land is devoted to corn for ethanol rather than corn for food. However the situation is more complex and in fact also depends on oil prices (Solomon, 2010). The % of corn that is used for food is the smallest of the corn uses at 10% and animal feed is the largest at 48%. Fuel takes up about 23% (Solomon, 2010).

    So if cellulosic material is so much better, why aren't we already using that? The commercial popularity of corn is because cornstarch is readily available, a relatively simple polysaccharide (figure 2) and therefore more easily converted to ethanol. Cornstarch also makes up 70-72% of the dry weight of a corn kernal. Lignocellulosic materials have a more complex structure and requires more enzymes and different types of micro-organisms to digest it;  because of this, they are not yet commercially viable. I will discuss this in my next post but for now, lets get back to ethanol and the types of micro-organisms that are used to convert it to ethanol.

    Ethanol producing micro-organisms
    Saccharomyces cerevisiae also known as budding yeast or bakers yeast is the micro-organism of choice in commercial bioethanol production (Figure 5).  Zymomonas mobilis  has also been heavily studied but S. cerevisiae remains top bug, mainly because Zymomonas mobilis need a specific subset of monosaccharides in order to produce ethanol where as S. cerevisiae is less of a picky eater (Bai et al, 2008). Conversion of corn to ethanol can be done through dry milling (67% of ethanol is produced this way) or by wet milling (33% ethanol is produced this way).  Bothast and Schlicher, 2005 give an excellent summary as follows
    Figure 5: Electron micrograph of S. cerevisiae

     "The wet milling process is more capital- and energy intensive, as the grain must first be separated into its components, including starch, fiber, gluten, and germ. The germ is removed from the kernel and corn oil is extracted from the germ. The remaining germ meal is added to fiber and the hull to form corn gluten feed. Gluten is also separated to become corn gluten meal, a high-protein animal feed. In the wet milling process, a starch solution is separated from the solids and fermentable sugars are produced from the starch. These sugars are fermented to ethanol. Wet mill facilities are true “biorefineries”, producing a number of high-value products. In the dry grind process, the clean corn is ground and mixed with water to form a mash. The mash is cooked and enzymes are added to convert starch to sugar. Then yeast is added to ferment the sugars, producing a mixture containing ethanol and solids. This mixture is then distilled and dehydrated to create fuel-grade ethanol. The solids remaining after distillation are dried to produce distillers’ dried grains with protein and are sold as animal feed supplement."

    It is in the fermentation process that S. cerevisiae uses the released glucose in the respiratory process of glycolysis. Remember back to your school biology? Glucose is broken down to pyruvate with the coincident production of the energy rich molecule ATP. Pyruvate can have many fates but one of them that occurs under anaerobic conditions is the production of ethanol. In animals cells lactic acid is produced instead of ethanol. But we have a problem. In both wet mill and dry grind conditions, harsh conditions are used - low pH and accumulating ethanol.  Glycoamylase, one of the enzymes that is used to converts starch into glucose works well in a pH of 4.5 and is continuously added during the fermentation process to ensure that all startch is converted to glucose. Now S. cerevisiae is one of the bugs I have personal experience with. Generally a neutral to slightly acidic pH (between pH 5-7) is best for growth and optimum temperatures were 30-37 degrees Celsius depending on the experiment. According to Bohast and Schlicher, 2005, fermentation occurs at 32 degress Celsius (no problem there) but the pH can sink to below 4. Ethanol concentration reach between 10-12% which is toxic and osmotic pressure is also a problem. How do the industrial yeast manage?

    Engineering yeast for biofuel production
    To enable the yeast to be at maximum efficiency for converting glucose to ethanol, many changes can be made to its genome. S. cerevisiae is a model organism, meaning that it is widely used as a model for studying genetic and protein interactions from which inferences can be made about similar genes and proteins in higher eukaryotic cells such as our own. The genetic manipulation of S. cerevisae is well studied and has a wide repertory of molecular techniques which are much easier to perform than those  necessary for mammalian cells. So, what has been done to allow S. cerevisiae to cope with high ethanol production (here's where it gets a bit more technical....)

    Increased tolerance to stress is a complex metabolic process and it is not usually the case that you can alter one gene and not affect anything else. Having said this, single gene manipulations have had some success. For example, increased expression of single amino acid transport systems have been shown to have protective effects and overexpression of the amino acid tryptophane permease genes (TRP1-5) have resulted in improved ethanol tolerance with little negative impact to growth. Other single manipulations of genes involved in uracil and galactose synthesis have also improved ethanol tolerance.

    Examples of other approaches that affect many genes are directed evolution and global transcription machinery engineering (gTME). In directed evolution, yeast is exposed to fermentation conditions, cells that survive are grown up and rexposed to fermentation conditions. Different batches are made testing for high ethanol and heat resistance for example. In this way, advantageous mutations accumulate. Increases in ethanol tolerance of 62 fold and heat tolerance of 89 fold have been reported (Zhao and Bai, 2009). gTME is the introduction of a randomly mutated copy of a transcription factor that controls transcription of a large set of genes. The transcription factor will then alter the transcriptional controls  depending on where in the genome it is introduced. Subjecting the engineered yeast to fermentation conditions will result in the survival of those yeast cells with improved ethanol tolerance. For example - 69% more ethanol production has been reported on a laboratory scale using this method (Zhao and Bai, 2009). 

    Another rather impressive aspect is the control of flocculation. Flocculation is the tendency of yeast to clump together. Self flocuclation is controlled by expression of certain genes and, by varying the stirring speed in the fermentor, the size of the floc can be also be controlled. Zhao and Bai, 2009 report on a study where the optimal floc size was found to be 300 um (micrometers) producing the highest cell viability when subjected to ethanol shock with concomitant improved ethanol yield. 

    These are just some examples of how the yeast genetics can be manipulated to improve ethanol yield and you can see how industrial yeast  ends up being very different from the yeast used to brew beer or bake bread and also demonstrates the flexibility of this micro-organism. 

    Here, I have talked about the use of corn as a feedstock for bioethanol and we have seen that it is likely not a long term solution to our energy woes. Several second generation biofuels are now being made from other feedstocks and with other micro-organisms. The use of corn is only the tip of the iceberg in terms of what is possible for the environmentally friendly production of fuel. Wait up for my next post to read about this!

    Definition of octane number: Octane number (or rating) is a value used to indicate the resistance of a motor fuel to knock. Octane numbers are based on a scale on which isooctane is 100 (minimal knock) and heptane is 0 (bad knock).  
    Examples: A gasoline with an octane number of 92 has the same knock as a mixture of 92% isooctane and 8% heptane.
    Knock: compressed gasoline-air mixtures have a tendency to ignite prematurely rather than burning smoothly. This creates engine knock, a characteristic rattling or pinging sound in one or more cylinders

    Bai, F. W., W. A. Anderson and M. Moo-Young (2008). "Ethanol fermentation technologies from sugar and starch feedstocks." Biotechnol Adv 26(1): 89-105.
    Bothast, R. J. and M. A. Schlicher (2005). "Biotechnological processes for conversion of corn into ethanol." Appl Microbiol Biotechnol 67(1): 19-25.
    Solomon, B. D. (2010). "Biofuels and sustainability." Ann N Y Acad Sci 1185: 119-134.
    Zhao, X. Q. and F. W. Bai (2009). "Mechanisms of yeast stress tolerance and its manipulation for efficient fuel ethanol production." J Biotechnol 144(1): 23-30.