Goo : Food and energy biotechnology is a very complex area. The biggest problem is knowing what needs to be done. I think the human race has gone up a few dry creek beds already in this area of expertise.
Kinkajou : The mandated inclusion of ethanol into petroleum fuels for vehicles is perhaps one such example. While we can produce ethanol from biological sources through farming, we are reducing food production to do so.
Goo : Perhaps we need to rethink what we have decided to do. It is possible that many things we have chosen to do are not worthwhile doing.
Kinkajou : True! Our goal of incorporating ethanol into petroleum fuels made sense in the context of a world oil crisis. It makes less sense to incorporate ethanol into petroleum fuels where world oil shale reserves are likely to supply our needs predictably for up to the next 100 years.
Still ethanol is a very good fuel and if appropriate source feedstocks such as plant-based lignocellulose are utilised in the production of ethanol, ethanol becomes a sustainably produced and commercially viable biofuel. We also need to plan for more than just a hundred years into the future. Learning what to do and how to do it takes time. The hundred years will speed into the past all too fast.
Bioethanol Feedstock Chemical Industry
Goo :So what are your thoughts Erasmus, on food and energy biotechnology?
Erasmus : The basic aim of food and energy bio- technology is the production of “molecules” for purposes of usage as “fuel” or for “construction”. (Construction being the use of molecules as basic building blocks for other molecules).
Food and energy bio technology is a Critical survival technology to the human race. Essential “Key Achievements” in this technical area could be summarised as:
- the production of alcohol (alcohol being a fuel predominantly) and
- The production of glucose, amino acids / proteins, oils or other bio-molecular building blocks.
Amino Acid Biomolecule Feedstock
Cytosine Nucleoside Biomolecule Feedstock
Kinkajou : Currently we have indeed achieved the capability to run many of these industrial processes.
We have developed extensive expertise in using petrochemical feedstocks for biological molecule production. Our current industrial techniques include the knowledge to convert petrochemical derivatives obtained by mining processes into the chemicals /monomers that are suitable for industrial processes.
Currently, we do not in industrial commercial capacity transform petrochemical products such as may be obtained by mining, into population wide quantities of “foodstuffs”. However, such a task is possible if not profitable or desirable at this time.
We also tend not to direct plant bio-feedstocks into biological molecule production.
It is the petrochemical mining industry that supplies the feedstocks for many of our industrial biological processes.
Petroleum Extraction Biomolecule Feedstock
Erasmus : True. I agree that what we have identified as essential, we in fact already have the capability to do. However, in the long-term these molecules must be produced “sustainably”.
These molecules must also be produced profitably. This means new processes need to be developed using new feedstocks. This means biotech molecules need to be derived from long-term sustainable business and commercial practices. We cannot mine petrochemical feedstocks for our industrial processes forever.
Imagine where we will be hundred years from now. There could well be 20 to 30 billion people on the planet. Dr Axxxx mentioned 70 Billion. Petrochemical Oil is becoming a very expensive commodity. Sustainable biotechnology is about renewable inputs, and recognising the relationship between cost and yield (i.e. efficiency).
Kinkajou : Yes, we can do it but we need to do it cheaper and better and more sustainably.
Erasmus : Exactly!
Kinkajou: So Erasmus, what do you think we should be aiming to achieve.
Food Biotech > Feedstock Biotech
I think the necessary achievements for humanity in the Food / Fuel Biotech arena are best summarised as:
- developing better methods for the use of plant materials as feedstocks for industrial processes
- synthesizing, storing and extracting glucose for uses as food for people or as biofuel for bacterial molecular synthetic processes
- developing better methods for the conversion of sunlight, carbon dioxide, water, and minerals into molecular biopharmaceutical chemicals
- Developing methods with “rapid” ramp up characteristics for the production of molecular bio-chemicals: (In the event of a crisis, food production is the most obvious rate limited industrial process in the world today).
There must exist methods for the rapid acceleration of production of bio-molecular “food” bio-chemicals. This could involve the conversion of petroleum feedstocks into food protein or oil (e.g. something like the Manna mentioned in the Bible), or the use of new high efficiency food production techniques such as chlorella harvesting, or the simple production of glucose from waste biomaterials.
- Finally, we need to develop molecules suitable for fuel or energy uses.
Glucose Carbohydrate Biomolecule Feedstock
Fatty Acid Biomolecule Feedstock
Kinkajou : If plants are all made of cellulose and cellulose is really just a glucose polymer, harvesting glucose from plants should be really easy. So what’s the problem?
Erasmus: As usual the devil is in the detail. Plants are not just made of cellulose. There are other chemical compounds besides cellulose present. Also, while cellulose is composed of glucose, the three-dimensional structure of the glucose is extremely important, in that it dictates the energy content of the individual glucose molecules in the cellulose.
Cellulose is extremely hard to digest, due to the tight crystalloid packing of the glucopyranose/ glucose molecules and the low energy state of the glucose molecules resident within the cellulose structure. Although the glucose molecule is a basic ingredient of cellulose there are energy differences between simple glucose and the varied glucopyranose structures.
Extracting glucose from cellulose takes time, energy and skill.
Fate Plant Biomolecule Feedstock
Goo :So what are plants actually made of?
Erasmus : The main structural molecules present in plants can be described as: cellulose (which is a linear polymer made up of glucose (D-glucopyranose) units, hemicellulose and lignocellulose. These monomers/ / polymers are combined in very specific ways giving a complex and stable structure.
The glucose (D-glucopyranose) units are combined in a linear or chain like structure. These aggregates vary in length and width and cross-linking. As a result, these elementary fibrils form ordered or crystalline sections and less ordered more amorphous regions.
Several of these elementary fibrils with an average thickness of about 3 to 4 nm, typically associate with each other and are then held together by a monolayer of hemicelluloses, generating 25 nm wide threadlike structures which are enclosed in a matrix of hemicellulose and proto-lignin. This natural bio-composite would typically be described as a cellulose microfibril.
Plant Structure Lignin Cellulose
Note the extra carbon atom outside the six ring of 5 Carbon atoms +1 oxygen atom. It is a six unit ring in pyranose made of 5 carbon atoms out of the 6 carbon atoms of the glucose skeleton.
Goo : Confusing and Complex!
Dr Xxxxx : Cellulose is actually a linear polymer made up of glucose (D-glucopyranose) units linked together by -(1-4) glycosidic bonds (-D-glucan). A structural and conformational analysis of cellulose indicates that cellobiose (4-O--D-glucopyranosyl--D-glucopyranose) rather than glucose is its basic structural unit4.
Pyranose is a collective term for carbohydrates that have a chemical structure that includes a six-membered ring consisting of five carbon atoms and one oxygen atom. There may be other carbons external to the ring. The pyranose ring is puckered, to allow all of the carbon atoms of the ring to have close to the ideal tetrahedral geometry. This puckering leads to a total of 38 distinct basic pyranose conformations: 2 chairs, 6 boats, 6 skew-boats, 12 half-chairs, and 12 envelopes.
These glucose molecules have conformational and stereochemical arrangements specific to the pyranose ring. They can interconvert with one another. However, each form may have very different relative energy, so a significant barrier to interconversion may be present.
Conformations of beta-D-glucopyranose
Relative energy of beta-D-glucopyranose conformers
Beta D Glucan Conformations
Energy Beta D Glucan Conformations
Kinkajou : You also mention something called lignocellulose. So tell us Erasmus, what is lignocellulose?
Erasmus : The term lignocellulose includes a range of plant molecules/biomass containing cellulose, with varying molecular chain length, and varying degrees of polymerization as well varying amounts of lignin. In plant tissues, hemicelluloses are generally combined with lignin.
Dr Xxxxx : Lignin is a phenolic macromolecule that is primarily formed by the free-radical polymerisation of p-hydroxy cinnamyl alcohol units with varying methoxyl contents.
The chemical structure of lignin is very complicated and is based on three monomeric precursors: coniferyl alcohol (with an aromatic (phenolic) group of “guaiacyl”, sinapyl alcohol, (with an aromatic (phenolic) group of syringyl and p-coumaryl alcohol, (with an aromatic (phenolic) group of p-hydroxybenzyl. The proportion of these monomers varies among species of plants.
The most important physical property of this organic macromolecule is its rigidity, which not only gives strength to the plant tissue but also prevents the collapse of the water-conducting elements.
Softwood lignins are almost exclusively composed of residues derived from coniferyl alcohol (lignin type G), whereas hardwood lignins contain residues derived from both coniferyl and sinapyl alcohols (lignin type GS).
In contrast, lignins derived from grasses and herbaceous crops contain the three basic precursors (lignin type HGS).
As a consequence, hardwood lignins have higher methoxyl content, are less condensed and are more amenable to chemical conversion than lignins derived from softwood such as conifers.
Kinkajou : So tell us about that last constituent of plants that you mentioned, namely hemicellulose.
Erasmus : Hemicelluloses are plant hetero-polysaccharides whose chemical nature varies from tissue to tissue and from species to species. These polysaccharides are formed by a wide variety of building blocks including pentoses (e.g., xylose, rhamnose and arabinose), hexoses (e.g., glucose, mannose and galactose) and uronic acids (e.g., 4-O-methyl-glucuronic and galacturonic acids)
Dr Xxxxx : Generally, they fall into four classes: (a) unbranched chains such as (1-4)-linked xylans or mannans; (b) helical chains such as (1-3)-linked xylans; (c) branched chains such as (1-4)-linked galactoglucomannans; and (d) pectic substances such as polyrhamnogalacturonans. Some hemicelluloses, particularly heteroxylans, also show a considerable degree of acetylation.
Hemicelluloses are generally rod-shaped inn structure with branches and side chains folded back to the main chain by means of hydrogen bonding. This rodlike structure facilitates their interaction with cellulose, resulting in a tight association that gives great stability to the aggregate.
The hemicellulose content of softwoods and hardwoods differ significantly. Hardwood hemicelluloses are mostly composed of highly acetylated heteroxylans, generally classified as 4-O-methyl glucuronoxylans. Hexosans are also present but in very low amounts as glucomannans. Owing to their acidic characteristics and chemical properties, hardwood xylans are relatively labile to acid hydrolysis and may undergo auto-hydrolysis under relatively mild conditions.
In contrast, softwoods have a higher proportion of partly acetylated glucomannans and galactoglucomannans, and xylans correspond to only a small fraction of their total hemicellulose content. As a result, softwood hemicelluloses (mostly hexosans) are more resistant to acid hydrolysis than hardwood hemicelluloses (mostly pentosans).
Erasmus : we can show some of these facts diagrammatically.
PRETREATMENT OF LIGNOCELLULOSE
Erasmus: Pre-treatment of the lignocellulose feedstocks comprising cellulose hemicellulose and lignocellulose is critical to enable the glucose built into the materials to be accessed. This process is often referred to as Saccharification.
Small particles need to be created by milling processes perhaps analogous to people using teeth to chew food prior to digestion. Physical pre-treatments, such as milling and microwave irradiation, have also been utilised to enhance the hydrolyzability of lignocellulosic materials. However, the major disadvantage of these methods is the high energy requirement.
Milling generally results in a reduction of substrate particle size (increases the available surface area) and a decrease in cellulose crystallinity and degree of polymerisation. Various kinds of mills have been evaluated, such as ball, hammer and two-roll mills. Exposure of cellulosic residues such as sugarcane bagasse to gamma radiation also resulted in a substantial decrease in the degree of polymerisation of cellulose but with only a marginal increase in substrate hydrolysis.
The ease with which starch substrates are hydrolysed can be increased by milling, which enhances swelling and increases the available surface area of the substrate. Lignocellulosics, however, require more drastic measures to increase accessibility because they have been primarily designed by nature to act as structural materials.
In order to make pre-treatment an economically competitive process, the method must also result in high recovery yields of hemicelluloses and lignin for further utilization as chemical feedstocks
Chemical pre-treatments tend to solubilize hemicellulose and lignin in order to expose the cellulose component to acid and/or enzymatic hydrolysis. A wide variety of chemicals have been suggested in the literature and these include sodium hydroxide, sulphur dioxide, aqueous ammonia, calcium hydroxide plus calcium carbonate, phosphoric acid, alkaline hydrogen peroxide, inorganic salts with acidic properties, ammonium salts, Lewis acids and organic acid anhydrides, acetic acid, formic acid, sulphuric acid, n-butylamine, n-propylamine and alcohols (methanol, ethanol or butanol) in the presence of an acid or alkaline catalyst.
Combinations of methods can be very effective such as when, a considerable improvement in the hydrolysis of wheat straw was obtained when gamma radiolysis was used in the presence of dilute sulphuric acid.
The micro particles need to be prepared for digestion either by acid hydrolysis or through the use of steam “cracking” techniques. Ammonia fibre expansion, organo-solvent treatment, sulphite pre-treatment, (SO2-ethanol-water) fractionation, alkaline wet oxidation and ozone pre-treatment have all been used.
Erasmus : In this regard, dilute acid hydrolysis has been investigated using a wide range of catalysts such as hydrogen fluoride, sulphuric acid, nitric acid, and hydrochloric acid. However, when dilute acid hydrolysis was evaluated at a commercial scale, sugar degradation was found to be high. Humic substances which were inhibitory to fermentation were produced and other operating problems, such as acid corrosion and the need of extensive effluent treatment, were sufficient to call the efficiency or desirability of these processes into question.
(Humate is a common term used to describe dry mined carbon based materials resulting from plant decay as may be typically seen near coal deposits. They are correctly called Leonardites or oxidized Lignites.
Even though pre-treatment by acid hydrolysis is probably the oldest and most studied pre-treatment technique, it produces several potent inhibitors including furfural and hydroxymethyl furfural (HMF) which are by far regarded as the most toxic inhibitors present in lignocellulosic hydrolysate. The presence of inhibitors will not only further complicate the ethanol production but also increase the cost of production due to entailed detoxification steps.
Enzymatic processes can then be applied either through biofilms, insertion of enzyme into the feedstock substrate, or through the application of biological organisms.
Successful saccharification of cellulosic residues has also been accomplished using highly specific enzymes.
However, efficient enzymatic hydrolysis requires some form of pre-treatment to open up the structure of lignocellulosics. Even starch requires some form of pre-treatment to enhance its rate and efficiency of hydrolysis.
Biological pre-treatments result in partial delignification of lignocellulosics using lignin-degrading microorganisms such as fungi and bacteria. Reductions up to 65% in the lignin content of cotton straw have been reported more than two decades ago using white-rot fungi.
However, lignin biodegradation is a very slow process that can be considered cost effective only if applied in conjunction to other physical and/or chemical methods such as thermo-mechanical pulping and steam explosion. In both cases, removal of resins and other extractable materials can also have an important role in improving accessibility of lignocellulosics to (bio) conversion.
Improving Yield is the essential goal.
- Most pre-treatment processes are not effective when applied to feedstocks with high lignin content, such as forest biomass. Due to the complex nature of the carbohydrates in lignocellulosic biomass, a significant amount of xylose and arabinose 5-carbon sugars derived from the hemicellulose portion of the lignocellulose) is also present in the hydrolysate.
For example, in the hydrolysate of corn stover approximately 30% of the total fermentable sugars are xylose. (Corn stover consists of the leaves and stalks of maize plants left in a field after harvest and consists of the residue: stalk; the leaf, husk, and cob remaining in the field following the harvest of cereal grain. Stover makes up about half of the yield of a crop and is similar to straw.)
As a result, the ability of the fermenting microorganisms to use the whole range of sugars available from the hydrolysate is vital to increase the economic competitiveness of cellulosic ethanol and potentially bio-based proteins.
In recent years, metabolic engineering microorganisms used in fuel ethanol production has shown significant progress. Besides Saccharomyces cerevisiae, microorganisms such as Zymomonas mobilis and Escherichia coli have been targeted through metabolic engineering for cellulosic ethanol production.
Recently, engineered yeasts have been described efficiently fermenting xylose, and arabinose, and even both together. Yeast cells are especially attractive for cellulosic ethanol processes because they have been used in biotechnology for hundreds of years, are tolerant to high ethanol and inhibitor concentrations and can grow at low pH values to reduce bacterial contamination.
Using Plant Feedstocks: Promise and Problems
Kinkajou : So let’s go back to your original statements, Erasmus.
You said that it was important to develop better methods for the use of plant materials as feedstocks for industrial processes
Erasmus : Yes it makes more sense to make better use of the feedstocks we have that we currently waste than to divert the use of arable farmland into growing corn or sugar cane to use as feedstock for yeast mediated fermentation.
Just think about it. We harvest the corn seed but the remainder of the corn stalk is often simply used as animal feed or just ploughed back into the ground. The corn stalk is actually a complex polymer composed of cellulose, hemicellulose and lignocellulose. If we can extract glucose from these materials, we have a ready made source of food for people, food for bacterial or yeast bio- factories and a ready-made substrate for other industrial processes.
There is technically more glucose available in the plant stalk then we harvest as seed from the plant, by a huge margin. If we can utilise the plant stalk is a bio-molecule source, we could potentially increase useful yields by at least a factor of 10.
There are some difficulties. It will be some time before we can make use of many different feedstocks from different sorts of plants due to the variability of composition of the lignocellulose in different plant cell walls.
Currently our industrial processes require a specific quality of grades of lignocellulose to enable these industrial processes to be run profitably and efficiently. Consequently, currently we have done substantial research into choosing suitable plants from which to derive our lignocellulose cellulose and hemicellulose feedstocks.
Maize Stover Preservation
Lignocellulose can be sourced from wood from forestry, short rotation coppice (SRC), and lignocellulosic energy crops, such as energy grasses and reeds. The use of forestry timbers harvested for biofuel production is not considered sustainable, however the use of wood waste material left over from other industrial processes is acceptable.
Lignocellulosic crops generally have a higher GHG (greenhouse gas) efficiency then rotational arable crops since they have lower input requirements and the energy yield per hectare is much higher.
Choice of Plants for Bioethanol Production
Kinkajou : So what other factors are important in choosing a plant source for cellulosic ethanol production?
The major factors affecting choice of source plant crop includes :The degree of yield per hectare, lignification extent and nature, and the ability of the plant to tolerate environmental stresses such as limited water, low nutrient levels, soil salinity, soil pH, and the ability of the plant to create residual organic matter within the soil layer. These factors vary from crop to crop, and amidst different species.
Different plant species may require different types of conversion process (e.g. acid and enzymatic hydrolysis, steam fracture and/or heating, or nano-milling pyrolysis. There is a role for plant breeding and plant selection to be used to optimise the traits of potential energy crops. Genetic research may also allow the identification of plant genes coding for improved processing of plant feedstocks. Gene technology may be important in achieving efficient cellulosic ethanol production.
Mallow a potential Biomolecule Feedstock
Kinkajou : Tell us about some of the different plants that have been considered as substrates for cellulosic ethanol production.
Erasmus : I’ll give you a list of some plants and their advantages and disadvantages when used for bio- molecule production
- Miscanthus (Miscanthus giganteus and other Miscanthus spp.)
Advantages rapid biomass accumulation in temperate climates, high yields double corn's low fertiliser requirement, adds significant amounts organic matter to soil up to 12 tons of biomass per acre to the soil (dry mass) over five years.
Giant reedgrass (Arundo donax)
Advantages adapted to a wide variety of ecological conditions but especially useful in wetland systems, minimal requirements on soil tillage, fertilizer and pesticide, protection against soil erosion, is well adapted to saline soils and saline water, and resistant to biotic and abiotic stresses. The fact that it can be cultivated for between 20 to 25 years without replanting
Reed canary grass (Phalaris arundinaces)
Advantages good yields and poor soils and contaminated land, useful for bioremediation of brownfield sites as well as a source of biomass for bioenergy (typically as briquettes) or pulp. Is also considered a suitable feedstock for cellulosic ethanol production [
Giant King Grass, Napier Grass, Elephant Grass (Pennisetum purpureum)
Advantages can be used as fodder plant, an attractive energy crop because it reaches yields up to 40 tons/ha/yr. and can be harvested 4–6 times a year, water and nutrient requirements are low. Possible feedstock for advanced biofuels, and bio-methane for energy production.
Switchgrass (Panicum virgatum)
Advantages wide climactic/geographic range tolerance, presence of seed suitable for market all further processing. Suitable for biofuels feedstock and genetically modifiable to grow with low amounts of lignin hence boosting biofuel yields by up to 33%.
Short Rotation Coppice of Willow and Poplar
(Salix spp. and Populus spp.)
Advantages Short Rotation Coppice SRC - where species such as willow and poplar are grown on marginal land typically over 3-5 year cycles and may be harvested annually over this cycle time - has potential for providing feedstocks for advanced biodiesel and drop-in biofuels (via thermochemical conversion) and for production of cellulosic ethanol (via biochemical conversion).
- Sugarcane: Sucrose (a dimer of glucose like monomer sugars) is obtained in as the main by-product of growth. Sugar is easy and well known in processing to ethanol.
Rusby or Virginia mallow (Sida hermaphrodita) member of the mallow family
Advantages Plantations can be used up to 25 years whereby the dry woody parts are harvested from late autumn to spring with high and stable yields. It is an easy to handle crop that can be cultivated with conventional farming methods and machinery.
Halophytes (Various species)
Halophytes as feedstock for bioethanol production were explored. These perennial grasses are salt tolerant with high growth rates and produce lignocellulosic biomass of "good" quality as a feedstock Species such as Halopyrum mucronatum, Desmostachya bipinnata, Phragmites karka, Typha domingensis and Panicum turgidum, have potential as bio-ethanol crops.
'Straw plants' that are easier to break down but are not otherwise weaker or smaller.
Variants of the model grass species Brachypodium
- Sweet Sorghum: Stalk or grain
- Cassava 5,549
Sorghum Biomolecule Feedstock
Cassava Biomolecule Feedstock