Biofuels and synthetic biology

How synthetic biology has influenced research in the production of clean energy in the form of biofuels

Algal Biodiesel: a hope for a greener, cleaner and more economical energy alternative

Most of the fuel we use today -whether it is kerosene to cook food or the petrol/diesel we use in our cars are sourced from petroleum products. Petroleum products are non-renewable -they are not usable in the long term- as they don’t get replenished at the source upon consumption. Also, most fuels contribute greatly to pollution, global warming and climate change in the long term.

Such deleterious changes in the global living conditions coupled with an ever-increasing demand for fuel and the shortage of petroleum reserves calls for alternative sources of energy which are easily available, affordable and environment friendly. Thus, in response to this demand, a lot of research is being focused on the development of sustainable fuels which can meet these parameters. One such area where a lot of work is being put in is in the development of biofuels.

A biofuel(i.e. a biological fuel) is any fuel obtained from the processing of material from the plant, animal or microbial sources. It is certainly a cleaner alternative to petroleum-based fuels; as carbon dioxide emissions into the atmosphere considerably go down; thus potentially helping in reducing global warming. Also, these fuels burn more efficiently than traditional fuels, as a result of which there are fewer nitrates, sulphates and particulate matter released into the atmosphere and hence the pollution causing ability of these fuels is considerably lower.

Types of Biofuels

Biofuels are of three major types based on the source of extraction:

1. Cellulosic biofuels: Cellulose sources such as wood chips, crop residues, bagasse etc. are pretreated and microbes are allowed to grow on them. These microbes use enzymes like cellulase to imbibe sugars into their system, which under the right conditions can form alcohol; which is mixed with conventional vehicular fuels to make engine emissions safer for the health of both the environment and humans. Corn is a common source of alcohol for fuels.
2. Biomass-based diesel: Also known as biodiesel, this is extracted from animal fat, leftover oil from restaurants or even algae with the larger cell membranes. This oil or fat is mixed with alcohol and processed to produce biodiesel.
3. Advanced biofuels: Another kind of biofuels generated from sources other than alcohol; for example, chemicals called isoprenoids(analogues of the simplest units of natural rubber), which are sourced from both plants and animals.

Synthetic biology in biofuel production

Micro-organisms(mainly bacteria) play an important role in biofuel production. They help in processing the raw material, technically known as feedstock, into products of use. The raw material is typically a complex substance obtained from plant or animal matter, which is broken down into simpler substances by these microbes. The microbes take in these simple substances; process(metabolise) them and under the right conditions, release the product substance(which is either alcohol or perhaps anything else). This product is now extracted, purified, processed and turned into usable biofuel. This is typically how biofuel is made.

Now, to produce a good output of product in terms of quality as well as quantity, we modify these organisms genetically by introducing new pathways to do so. The whole procedure by which this is carried out is known as Synthetic Biology. Synthetic Biology uses a “toolbox” of genetic and catalytic parts -which can be fit together like a jigsaw puzzle to give us a product of our choice. Once a thorough study of what we want is carried out; a basic design for the desired pathway is created by genetic modification of an individual called a host(which is bacteria most of the time), to form an artificially created individual called a clone. The product is tested for efficiency and if necessary, minor code modifications are carried out on the clone to ensure an optimised outcome as far as yield and productivity of the product are concerned. All this is to ensure that new methods of biofuel production are created, and existing ones are upgraded.

The research in the domain of biofuel production enhancement is happening through four approaches:

Traditional fermentation:

The main objective of the traditional fermentation method is to enhance the production of ethanol, isopropanol and butanol, the three alcohols produced directly in nature. Isopropanol and 1-Butanol produced traditionally using a species of bacteria called Clostridium resulted in 20 and 2 grams per litre respectively. Another approach was taken, where a gene which coded for a pathway giving acetone as a product was inserted, along with one which encoded alcohol dehydrogenase(ADH) enzyme into a bacteria called E.coli. When both genes were put in action, the transformed E. coli produced isopropanol by reduction of the acetone by this ADH; in higher quantities than that produced from Clostridium. However, the production of 1-Butanol by a similar mechanism turns out to be a bit difficult; as the yield of 1-Butanol turns out to be very less(o.o5 grams per litre, as opposed to the traditional method’s 2 grams per litre), perhaps due to enzyme activity being low as a result of the gene not producing proper protein, oxygen sensitivity or even due to the enzyme getting dissolved in the cellular medium, which is highly undesirable. Work needs to be done as far as the yield of 1-butanol is concerned.

A Scanning Electron Micrograph(SEM) of E. Coli

Non-fermentative higher alcohols

We produce alcohols which are otherwise not produced naturally in the host. But, we can’t just create a genetic pathway and insert it into them, as it may end up adversely affecting the health of the host. To prevent this, the metabolic reactions the host undergoes is taken advantage of. The product interest from these reactions is partly utilised as precursors for producing new kinds of alcohols using enzymes whose genes have been introduced by into the host from a new host. For example -the gene an enzyme from the bacteria Lactococcus (the kind of bacteria used in cheese production) which is used in Lactic acid breakdown, was inserted into to E. coli bacteria. Similar substances, which are respiratory products of E. coli, interact with this enzyme to eventually form six different alcohols, which are larger and not naturally produced, but useful in biodiesel production. Isobutanol production surpassed 20 grams/litre(g/L)- thus very high produce was obtained. Longer alcohols are, in fact, advantageous in producing biofuels as they don’t mix with water very well, and as a result, can be easily separated from the medium in which our clones are growing. Other bacterial species have also been used as clones to produce different kinds of alcohols as well. The yields have been in the range of 2.8 g/L to 9.5 g/L; which is better than many traditional alcohol production methods.

Lipids

Here is something very different we want to do. Instead of using alcohol produced from these clones; we use the fat present on the cell membranes of the host- we extract it and combine it with alcohol instead of the other way around. Fat is produced in a cell from a substance called Acetyl-CoA. Sometimes, genes of enzymes involved in fat synthesis and that of enzymes involved in ethanol pathways are cloned into the same host. This leads to the production of a compound called Fatty acid Ethyl esters(FAEE), which are sources of biofuel. The range of yield of FAEE in different researches has been between 12 mg/L to about 4.5g/L; thus indicating a need for improvement here.

Direct incorporation of Carbon Dioxide:

Carbon dioxide-based technologies are used to produce precursors to alcohols, and even alcohols themselves. This method is used in photosynthetic algae and cyanobacteria(a kind of photosynthetic bacteria). Here, the biochemical pathway governing photosynthesis directly or indirectly linked to the respiratory cycle, where the production of alcohols or precursors as mentioned in the second method takes place. One interesting substrate that can be used is isoprene analogues- isoprenoids(a modified version of the simplest unit of natural rubber). A gene taken from the kudzu vine coding for something called isoprene synthetase is taken to produce isoprenoids from one of these analogues. The isoprene synthetase is programmed to be light controlled; so that we can control isoprene production as per our needs. This isoprene is eventually converted to isopentanol. The yield for this is pretty low- i.e. 723mg/litre to 1.2 g/litre. This is pretty low yield, and hence a lot of research is being done to optimise this; as this sequestrates carbon from the environment, thus giving this method an edge in terms of its eco-friendliness.

Challenges of using synthetic biology methods

Despite the progress being made in this domain of research, there are still huge challenges to be overcome to allow these methods of biofuel production to reach their full potential:

1. Finding ways to keep the metabolic strain on the hosts and clones while incorporating foreign genes and producing foreign products using the same and ensuring efficient consumption of nutrient medium to produce usable amounts of product.
2. Production methods must be better. Proper maintenance of environmental conditions- with the right temperature, pH, amount of carbon dioxide, oxygen and nutrient balance.
3. Progress in optimising production of the desired product by enhancing gene expression, while not adversely affecting the metabolic pathways which are essential for the survival of the host.

Future scope

1. With technological advancements, research tools are becoming more and more precise and allow a deeper study at fundamental levels of anything. So, a broader study of metabolic processes exploitable for the job can be done, and genes can be designed accordingly. This would be helpful in a way that there would be a purer product produced with a lesser impact of the organism’s metabolic reactions on it.
2. Synthesis of whole independent genomes which can replace existential genomes can also be considered a thing of the future.
3. A larger “toolbox”, to allow a larger array of hosts to be accommodated and used.
4. These changes can not only help in applications of synthetic biology in biofuel production but also fields like agriculture, healthcare and many other sectors

Overall, research on biofuel development with the help of synthetic biology is moving in the right direction. A lot of effort is being put into optimising and increasing the productivity of the microbial sourced substances. Integrating these methods into the mainstream completely is just a matter of time.