Milking the microbes

The use of synthetic biology techniques is opening new doors in the production of renewable biofuels but, as E&T discovers, this work is still at an early stage of development.

Until recently, biofuels had been a much heralded answer to our growing CO2 emissions from transport. That was until controversy erupted over their use of feedstocks that are needed to feed the spiralling world population.

Using food such as wheat, corn or sugar, the so-called first-generation biofuels were always struggling for acceptance despite the fact that they can reduce CO2 emissions by up to 65 per cent. Second-generation fuels rely on the extraction of fuel from the non-food parts of crops that remain once the food crop has been extracted, as well as other crops that are not used for food purposes, while third-generation processes are being developed where algae are farmed for their fuel content.

But the drive is now on to develop fuels that are renewable, economical and do not harm the food chain. The answer appears to be fourth-generation fuels - or advanced fuels - where genes are modified to produce fuel, which they secrete, rather than having to be destroyed. These are 'living factories'.

Two of the leading exponents of the synthetic biology approach to producing fuels are California-based Amyris and Synthetic Genomics. Kinkead Reiling, co-founder of Amyris, says: "Traditional biofuels are ethanol, which goes into gasoline, and methyl esters, which are used to make diesels.

"These are products that are naturally found in nature and we have figured how to capitalise on that for transportation fuels - don't forget the original diesel engine was designed to run on peanut oil.

"For advanced biofuels - which as a name suggests is meant to be the next step, it is not just what we have found in nature, we are making some proactive steps to develop something that we feel is better."

He continues: "There are two sub-categories to advanced biofuels. There are advanced feedstocks; so new feedstocks that we previously couldn't use to make fuel - this is cellulostics. Then there are new fuels; things that are not ethanol, methyl ester biodiesels - we fall into that second category."

Yeasts and sugars

Reiling describes the microbes used in his company's process as yeasts that can eat lots of sugar sources. They can use the traditional feedstocks or the cellulostic feedstocks when they are ready. The fuels look like, and are designed to look like, traditional fuel, so they are truly a replacement, not an alternative. He explains that when people hear alternative, they think they have to change their engine and distribution network, but with a true replacement that entire infrastructure is useable for your new fuel.

"First of all, we identify the molecules in fuels that we would like to make," Reiling says. "We then identify where those molecules are naturally made. Now we know what we want to make, we go out and find some plants that make that compound.

"We take the genes from those plants and, using modern technology, we ask for the gene sequence and then can synthesise it and send it to you. Using specially designed systems we insert those into a microbe.

"The fuel that is produced naturally partitions out of the microbe. It goes out of the cell, through the cell wall and into the media; just like when you make salad dressing, the oil and water separate. Unlike ethanol, which is mixable with water and takes a lot of energy to distil, the oil produced by these microbes looks like oil so separates from water. To keep the process going, the microbes need a continuous source of fermentable sugar."

The process of synthesising the genes involves the use of recombinant DNA, a technique that has been around for some time. Dr Aristides Patrinos, president of Synthetic Genmomics explains: "You can only tweak a genome so much because you have a whole lot of machinery in the cell that is interested and involved in other things. Over the past few decades, we have been successful in tweaking various organisms to make them produce and convert what we require.

"We are convinced that to be cost effective and efficient, we ulimately need to start from a synthetic genome into which we programme all those functions that we are interested in.

"It is a case of starting from scratch and designing a genome that has only those functions that you are interested in, as opposed to having to carry the baggage that every living thing has because of its evolution and other priorities in life."

Algae farming

To achieve the advantages of advanced biofuels, the industry will need to transition from what you could call farming into a round-the-clock industrial production. Most of the work in algae is essentially farming - you grow the algae, you harvest it, you extract the lipids and then you grow another batch.

"We have managed in our metabolic engineering to tweak the genome of several algae in a way that can secrete carbon C8s and C10s," Patrinos explains. "They can secrete it into the medium and therefore move it continuously; I like to use the term that we are actually 'milking' the algae as opposed to crushing them , destroying them, and starting with a new batch."

The genetic engineering of algae requires - almost demands - the use of bio-reactors or contained systems so manufacturers can guarantee that these algae will not escape in the environment. That containment can be achieved either physically in a closed system, as well as biologically, by tweaking their genomes in a way that they could not survive outside the narrow conditions of the reactor environment.

Patrinos explains that sequencing alone is just the first step. "You get the A, T, C and Gs [adenine, thymine, cytosine and guanine] by themselves, but they don't really mean anything," he says. "What you have to do is translate these into all the functions that are represented.

"To discover the genes and figure out what they do in this symphony of life - which instrument each particular genome represents - you can do comparative analysis. If you discover the genes and their functions of one organism, when you look at other genomes you can infer the functions and make reasonable assumptions about what those functions will be and you short circuit the process. We call this 'annotating the genome' and it is a very important step in modern biology."

An analogy often made of the work of synthetic biologists is with that of a computer programmer. "What I would say is that, 20 years ago the way that you would do what we are doing now is to randomly look for microbes that happen to make what you want," Reiling says. "If you want to improve the process you would expose them to nutragens and, at some point, you would find one that happened to be better. It was a very luck-driven process - luck weighted with a lot of hard work.

"The model today is that we know the genome, we are able to selectively add, subtract or adjust functionality, and then we have rapid ways to obtain feedback to see if it had the desired effect. This is analogous to the idea of programme, compile, run, programme that you have for computers."

Marketable biofuels

Although most of the work to date is in the lab, both companies have had success with small scale marketable products. For Amyris, the product furthest along in development is its truck diesel.

"That fuel blends well and runs well in engines up to 50 per cent blends," Reiling explains. "It has a high cetane, which is an important characteristic so that you can blend with diesels. It has a very low gel point so that it can be used in the cold - you don't have the cold flow issues that you have with traditional diesels. It has a lot of advantages for blending."

It is being produced today, but not at the scale where it can be distributed broadly. The company plans to have a demonstration facility opened next year, a full-scale commercial plant the year after. Reiling adds: "We are working on scaling the process so that it works well in commercial applications and also optimising the process so that we can come to market at the lowest price possible."

Synthetic Genomics has a deal with BP to look at sub-surface hydrocarbons and, in particular, coal-bed methane production. Much of the methane that is produced from coal-beds is biogenic in mature.

By sampling it they have been able to understand the microbial environment that lives in the coal bed and, Patrinos says, they are quite confident that their understanding is sufficiently advanced that they can devise ways to stimulate the production of methane by these microbial communities.

He also points to another project in Malaysia with the Genting Group working on their oil palm and jatopa plantations. "We have sequenced the genomes of both of those plants and we are quite bullish about the opportunities we see for tweaking the genomes in ways that can make the production, the yield, of both plants much higher," he says. "We hope to change some of the properties so that they are easier to harvest.

"We have also been studying the microbial communities that reside in the root zone of these plants. We are very confident that the understanding of the processes involved in those risospheres will give us insights into ways by which we can devise microbial fertilisers which will reduce the dependence on petroleum-based fertilisers, as well as deal with some diseases that blight the oil palm plantations - a disease called Ganoderma, which we feel we have some pretty good tips for using microbial systems to contain that disease."

There are still challenges to be overcome before the processes can reach the stage of industrialisation. The challenges fall broadly in two areas: one is optimising the efficiency of the system; ensuring that as much of the input sugars goes to help the product and that the purification, or separation, captures as much of the fuel as possible at a low cost.

"Secondly, we need to industrialise the micro-organisms," Reiling adds. "There are certain rigours that you see in a manufacturing site that you don't see in the lab, so we have been moving our microbes to be able to handle the rough and tumble environment of commercial production."

One of these problems, he cites, is that sometimes tanks will be cleaned with acid so the microbes need to be pH tolerant. There are quite a few different hurdles, he adds, that a microbe has to get over to be viable at large scale in addition to make the fuel well, but purification is not one of them. "It has been surprisingly easy," he says.

"With ethanol, they have to distil it to get it out of the water, because ethanol is totally mixable with water and that takes a lot of energy. "Because the fuel looks like oil, it is nothing like as energy intensive to pull that away from water."

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