Ennetechby Erasmus and Kinkajou Authors

 

 

Erasmus and Kinkajou share their vision of technologies that will help us on our way.

Bio-Technology : Feedstocks for the Future

 

 

 

 

 

Microbes are small biomolecule factories with nanosized chemical and enzyme systems tuned to output specific bio-chemicals.

 

 

 

 

 

 

 

 

 

 

 

 

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What feedstocks Do we Need to Produce?

Cooking Lignocellulose  Biomolecule Feedstock

Kinkajou Kinkajou : Tell me why it is important to learn how to convert cellulose to glucose.

Erasmus Erasmus : Conversion of cellulose to glucose: This is a critically important step to ensure food supplies for the human race. Currently, there are many crises that can befall us. The trouble with food supplies, Is that the only way we can “make” more food is to grow it. This means we need to put in in the ground and then wait.

The more efficient the chemical processes are, in the long term the less land is needed to be diverted away from food production and into feedstock for fuel production.

There is a need to have an industrial process available to the human race which utilizes non-farmed feedstocks for food production. Having available to us a process such as the ability to convert bio feedstocks (petroleum) obtained by mining into a foodstuff such as the “manna” of the Bible, is important. The need addressed here is to have an industrial process which is independent of biological processes, able to produce food. This reduces our dependence on critical processes such as “growing” food stocks. In the event of a nuclear winter, the only food may be factory produced food.

This process of using an industrial process to produce food has been sort of mentioned in the bible. The manna mentioned in the Bible that was deposited on the ground would decay into a black sticky substance if not harvested.


Kinkajou Kinkajou : Well it does remind you of oil, even if they used some other feedstock for this chemical reaction.

Erasmus Erasmus : Yes I agree. However no one really knows what “manna” is.

 

Lignin Biomolecule Structure Lignin Biomolecule Structure

 

Kinkajou Kinkajou : So what other sort of molecular building blocks do we need to synthesise?

Erasmus Erasmus : We require other basic techniques for the improved bio-industrial production of a number of molecular building blocks. These have been identified as:

1,4 succinic, fumaric and malic acids
2,5 furan dicarboxylic acid
3 hydroxy propionic acid
aspartic acid
glucaric acid
glutamic acid
Itaconic acid
Levulinic acid
3-hydroxybutyrolactone
Glycerol
Sorbitol
Xylitol/arabinitol

Cell based biological processes are responsible for the majority of synthetic routes from plant feedstocks to building blocks. However chemical industrial processes predominated in transforming or converting these building blocks to molecular derivatives and intermediates used in industry. A Hundred years from now, using petrochemical feedstocks obtained by mining may not be cost efficient or sustainable on a planet suffering from the depredations of 20 to 30 billion people. Plant derived feedstocks may be the oil of the future.

 

Lignocellulose Processing protocols

Industrialization Of Food Production

Our Little Numbat Friend Goo : However, I note that there is little industrial or commercial production of foodstuffs for general consumption from petrochemical feedstocks.

Kinkajou Kinkajou : One of the major advances of the 20th century is the industrialization of food supplies. People in most western countries can enjoy apples and oranges all year round. Much of this provision of food is due to improved methods of food storage such as refrigeration and controlled ripening. Now many fruits can be stored for months and ripened with e.g. ethylene oxide.

Many greenies complain about our use of this tech. Still it means less wastage, cheaper food and the availability of these types of foods to more people for much longer. In the western world we are probably one of the first generations not to have faced hunger regularly.

Erasmus Erasmus : As we have already commented, food production is one of our slowest production processes. If we are to avoid starvation as human population of the planet increases, we need to double or triple food production in the next 100 years.  Learning how to store and preserve it better, is a technology of the past, not the future.

The availability of arable land is limited today. We are unlikely to expand the available area of arable land on the planet.

We have to use what we have better, smarter and more efficiently. We need to allow fallow space to be available for all the other life forms on the planet to prevent humanity from crowding out the other species existing. Our biodiversity is one of our most valuable but most unrecognised resources.

New Feedstocks for Food / Energy Production: Alcohol

We cannot afford to divert arable land from food production into usage for fuel production. If we use our land to make bioethanol for example, there is less land available to grow food.

Kinkajou Kinkajou : Hence, I see the key achievement is finding uses for biomaterials that currently are wasted. Similarly we need to improve efficiency of land use as well as reducing the wastage of arable land.

Erasmus Erasmus : let us look at the example of alcohol production. Alcohol has been produced since early this century by fermentation process based on yeast fermentation. Although capable of achieving industrial level outputs, the process is inherently inefficient. This is because alcohol production by yeast organisms occurs as a by-product of cellular growth.

The most efficient industrial processes is one whereby the cellular bio-factory receives controlled inputs, is unable to divert energy into growth ,but delivers specific outputs via steady state industrial process. Yeasts do not do this.

Kinkajou Kinkajou : Yes, yeasts produce alcohol as a by-product of the growth process. Consequently much of the energy input is diverted into the production of more yeast cells, not just into the production of ethanol for energy fuel.

 

Saccharomyces Yeast Saccharomyces Yeast

Erasmus Erasmus :
A new bio-organism based method of alcohol production is based on the bacteria Zymomonas mobilis . The most notable characteristics of this gram-negative bacterium is its bioethanol producing capacity.

The advantages of Z. mobilis over Saccharomyces cerevisiae (yeast) with regards to producing bioethanol include:

  • Limited cell growth and replication leading to lower biomass production
  • Higher tolerance for environmental ethanol up to 16% (yeasts tolerate only about 12%).
  • higher sugar uptake and alcohol yield
  • ability to tolerate a low oxygen environment during fermentation

Problems in the use of these bacteria for fermentation include:

  • The substrate feedstock range is limited to glucose, fructose and sucrose.
  • Substantially less tolerance for toxic inhibitors present in lignocellulose feedstocks such as acetic acid and various phenolic compounds.
  • Ability to utilise only a narrow range of metabolic processes, effectively limiting food stock inputs and biochemical outputs

KinkajouKinkajou : It seems strange that yeasts divert so much energy into growth.

ErasmusErasmus : Yes, I think we can draw some conclusions from the genetic structure of yeasts from this growth habit. The implication would be that much of the gene expression on the yeast cell DNA is regulated in a linear fashion. Initiation of DNA translation into protein products and mRNA occurs as the cell grows.

DNA translation starts at one end of the chromosome and just plods on till the end of the chromosome. Essentially all the DNA is utilised and expressed. Specific bits of DNA cannot be induced to have upregulated outputs depending on the circumstances affecting cell growth.

This unfortunately suggests that the DNA structure of yeasts is not suitable for the purposes for which we intend to use them for.
It seems to me that there needs to be a DNA operon with outputs that regulate cell growth at multiple points in the yeast cell DNA. This growth operon needs to be independently signalled and operated from operons controlling DNA expression of metabolic enzymes. With such an operon modified yeast cell, we can stop the cell from growing.


There needs to be specific operons controlling the up-regulation of accessory metabolic enzymes optimising the usage of lignocellulose feedstocks derived from farming. The yeast needs to adapt to its food source and needs to use available substrates efficiently.

 


KinkajouKinkajou : I can see the Zymomonas mobilis bacteria eventually undergoing extensive genetic modification into a range of subspecies, each subspecies being optimised for particular chemical processes. This seems easier than gene adapting yeasts for cellulose biofuel production. Depending on which bacterial subspecies is used in a chemical process, different bio-molecular chemical outputs can be achieved.

There is one other very important corollary of this type of genetic adaptation process. In having the bacterial factory fine-tuned to very specific chemical processes, it is unlikely that these bacteria can escape the laboratory situation to become a nuisance for a menace in other food or biochemical synthetic processes.

The last thing we need is to build a new gene modified super bacteria able to thrive in the brewing environment. If it is able to use a greater range of input substrates, to tolerate a greater temperature range, and to tolerate a greater range of toxic or environmental adverse factors,

it is much more likely to contaminate brewing processes or fermentative processes worldwide. A more robust germ that contaminates even 5% of brewing or fermentation runs would be a disaster.


Our Little Numbat Friend Goo : yes I agree. It seems to make more sense to adapt each sub strain of bacteria for a specific biochemical purpose, rather than to design super bacteria to achieve a range of purposes. Such an improved and versatile bacteria is much more likely to escape its intended biotech niche.

 

Fermentation Processing Fermentation Processing

Fermentation Processes In Food / Fuel Biotech

 

Kinkajou Kinkajou : Doesn't fermentation simply produce alcohol?
Erasmus Erasmus :
There are a number of different sorts of fermentation. Different metabolic fermentation processes have different outputs. The most important processes involved alcoholic fermentation and mixed acids fermentation metabolic techniques. Alcohol is as important as a fuel. However mixed acids fermentation is capable of producing a wide range of biochemical suitable for a number of industrial chemical processes.
The major types of fermentation are:

  • Alcohol fermentation (ethanol and CO2 are produced; using yeasts like Saccharomyces cerevisiae or bacteria such as Zymomonas mobilis ),
  • Homolactic fermentation (In this simple pathway, one glucose molecule generates 2 pyruvate molecules which are then converted directly to lactic acid; no gas is produced. This pathway  responsible for soured milk, in production of many dairy products:  germs used include: Lactobacillus, Streptococcus, Bacillus),

  • Heterolactic fermentation (In this process ethanol, CO2, and lactic acid are produced. Typical bacteria include : Leuconostoc and Lactobacillus),

  • Mixed acids fermentation (This is a complex anaerobic fermentive process where the products are a complex mixture of acids particularly lactic acid, acetic acid, formic acid and succinic acid as well as ethanol and quantities of hydrogen and carbon dioxide. While this fermentative process is characteristic of members of the Enterobacteriaceae family, other bacteria such as E. coli had the metabolic range to synthesise a wide range of bimolecular outputs.

    There is considerable variability in the quantity and identity of the biomolecules generated by this range of chemical processes. This does imply that there is considerable scope for optimization of genetically controlled metabolic or synthetic processes. This may become the metabolic process of the future for bacterial bio- factories we have yet to build.

  • Butanediol fermentation (Acetoin, a precursor for butanediol, is produced, for example by Enterobacter aerogenes).  It is interesting to note that butanediol is chemically related to GHB. Psychoactive chemicals can be produced just as easily as industrial ones.
  • Anaerobic butyric fermentation (organic solvents produced including butanol and butyric acid; Clostridium sp.),
  • Propionic acid fermentation (This is the fermentation type that makes holes in Swiss cheese, typical involved bacteria being for example Propionibacterium sp.).

Even the case of simple alcohol production, there are many choices to be made such as the nature of the fermentation microorganism. Different organisms are capable of utilising different substrate carbohydrates.

For example if starch and sugar are raw materials in the medium then specially selected strains of Saccharomyces cerevisiae are utilized. Production from Lactose of whey is accomplished with Candida pseudotropicalis. If it is sulphur waste liquor fermentation the Candida utilis is the best organism, because of its ability to ferment pentoses.

So particular strains of these various organisms actually employed for the fermentation are selected for several properties. Properties include growth speed, tolerance to substrate and alcohol concentrations temperature, pH and toxic lignocellulose by-product tolerance.

Fermentation Industrial Fermentation Industrial


Kinkajou Kinkajou: Do all these bacteria or yeasts use the same metabolic pathways?
Erasmus Erasmus : there are a number of metabolic pathways important in the production of alcohol, in fermentation, and the production of other metabolic by-products.
The main pathways involved in metabolism are:

  • Glycolysis: The most common type of glycolysis is the Embden–Meyerhof–Parnas (EMP pathway). This pathway is used for the large majority organisms.
  • The Entner–Doudoroff pathway describes an alternate series of reactions that catabolize glucose two pyruvate using a different set of enzymes .Most bacteria use glycolysis and the pentose phosphate pathway.

    Only prokaryotes use this metabolic pathway, most being aerobics, due to the low energy yield per glucose molecule requiring alternative energy sources to sustain cell growth and replication. They may lack enzymes essential for glycolysis, such as phosphofructokinase -1.

    • This pathway is generally found in pseudomonas, Rhizobium, Azotobacter Agrobacterium, Escherichia coli, Zymomonas mobilis, Xanthomonas campestris and a few other Gram-negative genera. Very few Gram-positive bacteria have this pathway, with enterococcus faecalis being a rare exception.
  • The pentose phosphate pathway.
    The pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) is a metabolic pathway parallel to glycolysis. While it does involve oxidation of glucose, its primary role is anabolic rather than catabolic. This pathway allows the production of C3, C4, C5 (ribose) and C7 sugars.

  • The primary results of the pathway are:
  • the production of energy via NADP used for example in reductive biosynthesis reactions within cells (e.g. fatty acid synthesis)
  • Production of ribose 5- phosphate, used in the synthesis of nucleotides and nucleic acids.

  • Production of erythrose 4-phosphate used in the synthesis of aromatic amino acids.
  • Dietary pentose sugars derived from the digestion of nucleic acids may be metabolized through the pentose phosphate pathway, and the carbon skeletons of dietary carbohydrates may be converted into glycolytic/gluconeogenic intermediates.
  • Conversion of sunlight to glucose or fats: This is a similar problem except that we don’t take indigestible cellulose as the feedstock for the industrial process to turn it into food. We use for example algae as little sunlight harvesting and storage factories. We select and modify a plant form that can maximise storage and minimise the diversion of photosynthesized chemicals into growth. Controlling growth is the key.

    To be useful we want minimal growth biomass and maximal foodstuff biomass. It would be interesting to see if we can   harvest: complex sugars like glycogen, fat or oils or proteins.
  • Conversion of glucose or other feedstock into alcohol. One method proposed has been the selection of bacteria that can take an input chemical such as glucose and metabolise it to alcohol. Currently, we use yeast as the main alcohol production organism. The trouble with yeast is that alcohol is really a by-product of the growth cycle. So it is impossible to separate alcohol production from growth.  This means that we routinely divert proportion of our energy from alcohol production to growth. This reduces yields and efficiencies.

    A solution to this problem is to use bacteria in steady state “starvation” mode. In this mode bacteria do not grow as they retain too little energy for growth. They do however continue to metabolise in steady state. This chemical process could generate clean green recyclable fuel production. But in the long term unless we can improve efficiency, the process produces a costly fuel. (With yeast as the conversion factory).

Metabolic Pathways for Synbthesis of Molecules
Metabolic Pathways for Synbthesis of Molecules

Review Critical Bio-Tech Processes

Kinkajou Kinkajou : So let’s review your list of critical biotech processes.
Kinkajou Kinkajou :

  • developing better methods for the use of plant materials as feedstocks for  industrial processes

Using plant materials as feedstocks for by industrial processes is a complex and difficult technique to master.

One issue is the variability of the structure of the plant feedstocks. Different physical chemical enzymatic or biological methods give different results on different substrates. A process optimised for one plant feedstock may be quite inappropriate for another. For example hardwoods are much more easily sacrificed using acid hydrolysis than softwoods.


There is a considerable role for the bio engineering of biological agents and enzyme systems to assist in processing of substrate feedstocks for the production of biological molecules.


I believe in the long-term enzyme-catalysed reactions will give the maximum energy and mass yield for feedstock processing.

Lignocellulose Biomolecule Feedstock
Lignocellulose Biomolecule Feedstock

  • synthesizing, storing and extracting glucose for uses as food for people or as biofuel for bacterial molecular synthetic processes

There are two issues in this area one is the actual processing of cellulose hemicellulose or cellular substrates to allow the production of glucose. The second issue is the partitioning of glucose from ongoing chemical reactions or within biological agents. For a molecular process to be commercially viable the glucose must be extracted and stored.

We have very little capacity at this point in time to gene engineer organisms for storage. Ideally our processing yeasts or bacteria would store glucose as for example an intermediate storage chemical such as glycogen which can be accessed later. This may involve killing the organism or initiating an export or transport reaction from the organism into the environment with glucose can be recovered.


In short our processing bacteria or yeasts need to store glucose in much the same way our bodies store fat.

  • developing better methods for the conversion of sunlight, carbon dioxide, water, and minerals into molecular biochemical

This group of processes looks at anabolic reactions such as synthesis based processes for the production of bio-molecular chemicals. Perhaps in the long-term using algae to produce glucose may make as much sense as using yeast or bacteria to extract glucose from cellulose.

The use for example chlorella algae has in the past been suggested as a valuable source for biological molecules or food production. Chlorella when dried gives a product consisting of approximately 45% protein, 20% fat, 20% carbohydrate, .5% fibre and .1% environment and minerals.

It was suggested as an expensive protein supplement to the human diet. The reality unfortunately exposed a number of processing issues which substantially reduced the value of chlorella is a food supplement. Chief among these is the difficulty in extracting the nutrients through a resistant and difficult to digest cellular membrane.

Other issues include maximizing yield, the requirement to grow chlorella in carbonated water which would substantially add to the cost of its production. Another issue is that perhaps the need to supplement light sources with artificial light, also adding substantially to the cost of production.
In the long-term, farming chlorella similar algae may well be a valuable source of calories or protein. 


However, modification chlorella or similar organisms may well be necessary to improve the accessibility and value of this food source. We have a long way to go.

  • Developing methods with “rapid” ramp up characteristics for the production of molecular bio-chemicals: (In the event of a crisis, food production being 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.

 

Food production is one of the most critical bottlenecks for the maintenance of human populations. To enable food security for the human population, there must be different ways of generating foods such as protein fats carbohydrates partners and minerals than by simple farming of plants. One method of achieving food security is the synthesis of foodstuffs from inorganic feedstocks such as petroleum chemicals obtained by mining.

Another suggestion is alternate synthetic pathway such as farming organisms like chlorella which have a much faster growth cycle than farm sourced crops. Another mechanism may well be the diversion of waste plant materials through processes which can generate glucose or other biomolecules.

Food security depends on choices and the biggest problem is the ability to make rapid choices, to choose processes which can be rapidly initiated and used to deliver by molecule outputs at a rate far in excess of direct farming techniques.

  • Finally, we need to develop molecules suitable for fuel or energy uses. In this example we have discussed the use of cellulosic ethanol production from waste plant feedstocks. These same molecules may also be required for the production of other intermediary biomolecules, able to feed into other industrial processes.

Kinkajou Kinkajou : Any Comment Goo?

Our Little Numbat Friend Goo : Food Good! Need Food!

 

What follows is a diagram showing how many commodity chemicals obtained from petrochemical feedstocks are converted into consumer goods such as textiles, food supply packaging, transportation accessories and solvents, housing products, clothing, recreational accessories, communications commodities, and health and hygiene accessories. The breadth and extent of understanding of chemical processes is breathtaking.



Uses of Biomolecules in Industry

 

 

 

Kinkajou Kinkajou : I really shouldn’t ask you these questions before lunch.

Our Little Numbat Friend Goo : Goo can see good thing when placed in front of his nose. If the human race ever achieves a population of 70 Billion, it will be the conversions of cellulose, in particular lignocellulose into glucose that will enable these people to be fed. What is amazing about this is that it could probably be done using less of a human footprint on the planet than we have now.

For example, when we harvest wheat or other grain crops, almost all the remainder of the plant goes to waste. If the entire crop were “food” (essentially grain or glucose from lignocellulose), I don’t doubt that the yield per hectare would increase tenfold.

The other important principle here is that the human race cannot continue using mined petrochemicals as industrial feedstocks forever. If it wants to continue as a technical civilisation, it needs to source its bio-molecules from a renewable source. I.e. it needs to grow these bio-chemicals, not just to dig them out of the ground.

Glucose Structure Image Glucose Structure Image
Glucose Structure conformation Glucose Structure Image