Archaea and Their Use in Making Biofuels
Archaea in the Ancient Past
From fossil evidence, scientists say that 3.5 billion years ago the oldest known living creature on earth appeared in its oceans. It was a bacterium, classified as a primitive prokaryote, a single celled organism. This origin of life, a single cell, was so simple it had no compartments and was just made of nucleic acids and proteins with a thin cell membrane, very much unlike most modern day bacteria. They were adapted to the environment then which was much different from today – it was a world with very little oxygen, and high ocean water temperatures among others. These ancient bacteria were first called archaebacteria (from the Greek word meaning ancient ones) and are generally believed to be the descendants of all living things on earth. They are still found today in harsh environments such as 2 km deep in oceans or in boiling springs such as in the Yellowstone National Park in the United States. (Priest,1994)
Archaea in recent times
Later scientific studies and findings showed that the term archaebacteria is not correct because these organisms are not really bacteria. Initially thought to belong to the same classification as bacteria , both being prokaryotes , archaea was later classified separately , thus the three kingdom domain that is recognized today is this : bacteria, archaea, and eukaryotes. Prokaryotes are one celled organisms that have no nuclei and other cell components, but with ribosomes RNA 70 S which makes them not sensitive to broad spectrum antibiotics. They also have and no peptidoglycan in their cell wall . Eukaryotes have nuclei, other cellular organelles, and ribosomes RNA 80 S. Algae, fungi and protozoa belong to this class.. These are one celled organisms without nuclei and other cellular structures, Their cell wall has no peptidoglycan, and they reproduce by binary fission. Nutritionally this class or organisms is so diverse they can be chemoorganotropic or chemoautotropic.
Further genomic studies in recent times verify that archaea may be the most ancient lineage of organisms, and that perhaps all further forms of life evolved from it.(Wang et al 2007). Originally archaea were thought to exist only in extremely harsh environments. Later they were found also in all habitats and it is now estimated to contribute up to 20% of total biomass on earth.
Archaea may be differentiated into 3 groups based on their physiology. However this classification is not a very strict one, as some members may exhibit common characteristics of two or more groups. This rough delineation is useful though as it provides an important information for ecological studies.
Halophiles which include the genus Halobacterium live in extremely salty environments with up to 25% salt or more. Thermophiles on the other hand can withstand very hot locations and some can even thrive in hot springs or inside volcano craters. If the organism has optimal growth at more than 80°C, it is called a hyperthermophile. There have been reports of organisms surviving up to 121°C. There are some thermophilic bacteria that can withstand high temperatures, but once the pH goes down to pH 5, only archaea are known to proliferate. Acidophiles are reported to withstand pH 0 and even lower .
In latest studies, it was discovered that archaea exist not only in mesophilic and thermophilic environments but surprisingly also in very low temperatures too. Methanogens of the archae family that produce methane gas, were found in cold sediments and other low temperature environments. Archaea in the world’s very cold oceans can come up to 40% of the microbial biomass. Because of their abundance in nature, and their presence since three billion years ago, more studies are needed to fully understand the physiology of these organisms and how they exert influence on the biogeochemical cycles of the oceans (interestingly, some marine archae were found to be able to nitrify, and this is a previously unheard trait of archaea so far.).
In the last decade, the biotechnology industry has shown a lot of interest in organisms of the Archaea Kingdom. As previously stated above, many of these ancient prokaryotes can live in some of the most hostile environments on the planet, such as hyper-saline lakes, acidic hot springs, and near-boiling deep ocean vents which have extreme environments of temperature, salinity, pressure, pH, radiation or oxygen tension. They can be found in volcanic hot springs, alkaline lakes, deep seas, oil wells, black smokers and highly acidic solutions that seep from ores and mine refuse piles. Even in extra harsh locations such as in mines 5 miles underground, having no light, water or air, in nuclear wastes, in geysers or beneath Antartica’s ice, amazingly, countless species of bacteria can still survive.
There is another type of archaea that produce methane under anaerobic conditions, and these are called methanogens.. They may also be found in wetlands . It is said that these organisms produce the methane found in flatulence. In marine sediments methane production by microorganisms happens below the top layers when sulfate substrates become too low.
In anaerobic environments methanogens remove products of anaerobic respiration. These could be hydrogen and other fermentation products. The gas methane that is produced is combustible and is also a biofuel. In areas beneath landfills and garbage waste areas where oxygen and other electron acceptors such as nitrates or sulfates are absent, methane gas is also formed by these archaea strains. Methane in large quantities can also be converted to methanol or even ethanol using specific catalytic reaction processes.
The term extremophiles is also used for the group of organisms of the archaea kingdom which have existed in such diverse and extremely harsh environments. Because of these special properties, they are important research subjects for ecological studies. They survived by developing unique strategies for stabilising their nucleic acids and other macromolecules in vivo, having new pathways of chemical transformation and energy transduction processes, and extremely heat-stable proteins( Ahring 2006).. Because of studies done on the proteins involved in the molecular adaptation of these organisms, scientists are now working on applications in industrial processes, one of the most important of which is on biofuels production.
All these varied characteristics of archaea led to a growing scientific interest in discovering their mechanisms of survival and how, thru gene transfer technology these traits can be transferred so they can help solve current energy and environment concerns of modern man.
Despite their potential, the use of extremophiles in production operations has so far not been as straightforward as hoped. It was found that when they are not in their natural habitat they can have very low cell yield. Understanding their physiological characteristics and optimal cultivation conditions is essential to obtain the highest possible yields and obtain their maximum potential.
Pressing Need for Biofuels
The great concern on global warming, the greenhouse effect and related climate based phenomena has come upon us in the last two decades. It is generally accepted that human activity that generates carbon dioxide and other greenhouse gases contributes to the problem. Energy requirements derived from fossil fuels such as crude oil or coal generates such environment sensitive gases. Thus the drive to turn to renewable energy sources such as the sun, the wind, and even waves. Plants are also a huge resource to provide biofuels that emit cleaner emissions, thus giving man another better option. Plants also absorb a lot of the greenhouse gas carbon dioxide during their growth period, thus helping decrease this gas in the atmosphere. Development of these alternative sources of energy, aside from resulting in a cleaner environment, has the added advantage of making man less dependent on finite fossil derived resources which are often imported and controlled by economic, geographic, logistic and political factors on a national level. Production of fuels and even other chemicals from locally grown plant material supports political and economic independence, a CO2 neutral energy production keeping dollars within the country. This is very important especially for remote and low income agricultural areas and developing countries. In addition to all these, fossil based energy sources are finite and are not expected to last long. Indeed there are several pressing reasons to look for alternative fuel sources if we are to act in a strategic way towards our world’s energy requirements for the next decades.
It is estimated that worldwide, about 27% of energy requirements is used for transportation. The segment is also growing at the highest rate. (Antoni 2007). This makes transportation fuels a good target to zero in on the direction to reduce greenhouse gas emissions. To date however, only a limited selection of energy-rich chemical compounds, including some liquid alcohols and esters, can be produced by microbes. This is because there are standards which have to be met regarding quality of the biofuels and strict compliance with the other criteria necessary for modern fuels are required. These include suitability for safe storage, capability to power transport engines and high energy efficiency in combustion engines. For air transport the energy density of the fuel also has high importance because the fuel comprises a significant fraction of the load of the aircraft. Much work still has to be done in this area to enable production of more types of fuels by microbes.
Biomass fuels have been used throughout man’s long history. Most of them were alcohols produced by the fermentation of substances like starch or sugars, others were plant oils. Aside from serving as fuel and energy sources they were also used as solvents, greases, cleaners or as basic chemicals. When the petrochemical industry was born, with its ability to supply these materials in quick and cheaper fashion, biomass fuels were forgotten. But now due to crude oil prices reaching heights of up to $100/barrel and increasing political instability in oil producing countries, the use of bio-based alcohols as solvents or basic chemicals is again under consideration. Add to this the fact that the amount of fossil derived fuels is quickly depleting, and estimated not to last more than 50 years.
Global Microbial Biofuel Production
The most important biofuels produced in high amounts are biodiesel and bioethanol. With far reaching potentials for the transport industry, bioethanol is produced using microbial fermentation. Almost 48.7 million m3/yr of bioethanol was produced worldwide in 2005, of which, 72.6% was produced in Brazil and the USA. The USA has increased its output in the past decade and is now the world’s highest ethanol producer. The market is further expected to expand in the next years as man becomes more conscious and enlightened about environment issues related to energy use. A rapidly expanding market for biofuels can be predicted for the near future. The demand of the EU25 countries for fuel ethanol is estimated to reach 11.4 million m3/yr in 2010, and a huge gap is foreseen between demand and production assuming a 5% displacement of conventional fuel on energy basis ( Antoni 2007).
As expected, countries allowing subsidies and tax exemptions for biofuels have the highest number of large manufacturing plants. Until the law of supply and demand catches up with biofuels technology, the industry needs to be subsidized initially until the time the business gets economically viable. It is worthwhile to note that where there is political will a country can go all the way with promoting biofuels for transport. Brazil for example claims already 50% of its vehicles are running on alcohol blends and they are targeting even higher blend combinations in the next years.
Obviously different plant species produce differing amounts of bioethanol. To date, sugar, starch and corn have been the usual substrates used in industrial production plants. As these are also food materials there is sometimes a source volume competition and prices are driven up by supply and demand considerations. Waste biomass would be a much better substrate so it has no competition with food.
Agriculture biomass such as leaves and stalks, of sugar cane , rice and corn and other crops can be used instead. This will even solve the problem of waste disposal if this biomass can be turned to bioethanol. In many countries agriculture waste biomass is just burned onsite, generating air pollutants and greenhouse gases, as the biomass on the field interferes with subsequent land preparation operations for the next cropping period after harvest. However, biomass has much higher lignin and cellulose content, and is much more difficult to decompose .These components have to be broken down to free the sugars which are the basic substrates of alcohol fermentation.
This is where microorganisms can help, by providing enzymes that can swiftly break up the original raw material substrate and speed up the decomposition reaction. It should be remembered that for all these processes, substrate production methods which do not interfere with the production of food must be developed, and further research needs on this area are numerous. The areas which will need microbial research were suggested as follows ( Antoni, 2007)
Utilization of cellulose and hemicellulose (whole plant) as substrate; new strains or
enzymes for biofuel fermentation, e.g. strains with higher fermentation temperature and
better product tolerance Improvement in energy balance including all relevant factors:
substrate, growth, refinery product separation, waste management and disposal,
It is foreseen that biofuels produced from renewable biomass of entire plants especially agriculture wastes such as straw or grass, are the sustainable energy resource with the greatest potential for ethanol biofuel production and microorganisms such as those of the archaea strain will have a big role in their future production increase.
Microbial biofuels have great development potential in process steps such as pretreatment, fermentation, substrate separation, energy coupling and others.
Biological research will need to contribute to an improved biofuel production by breeding of energy plants, enzymatic hydrolysis, specialised fermentation strains and waste treatment (Antoni 2007)
The Energy Challenge : Microbes to the Rescue!
Clearly the need for higher production volume for biofuels is a direction which has to be pursued if we have to meet the requirements of a growing transport industry which wants to be free of the shackles of dependence on the finite nature of fossil fuels, and wants to be less guilty of spewing our greenhouse gases. Microorganisms are up to the challenge as they could contain certain enzymes that can affect biofuel production processes.
This is where the archaea has recently come into focus. Studies have consistently shown that several strains of archaea release enzymes that can hasten bioethanol production. Indeed, what an irony of nature that such ancient nondescript microbes can help produce more biofuels in a more efficient manner, and aid in solving a modern day energy problem.
Using Archaea Metabolism to Help the Environment
Just like ordinary microbial reactions involving in vivo systems, the industrial process that is currently used to produce bioethanol from cellulose in plant materials is a slow, expensive and inefficient process. It is based on the fermentation process which is largely dependent on temperatures at which the catalyst works best, and unfortunately, sensitive to the presence of the product itself . This industrial batch fermentation procedure is costly and time consuming. The fermentation vessels need to be cooled for the catalytic reactions. The microorganisms are sensitive to ethanol, and once enough of the alcohol is produced, the organisms die.
The trick is to make huge vessels so that the ethanol produced is diluted, but at a certain concentration stage, the reaction has to be stopped. This is because the full industrial synthesis cannot typically proceed under the conditions necessary for recovery of product. Recovery is thus initiated after synthesis is complete. The ethanol is recovered in a separate vessel in another manufacturing step. There is thus frequent downtime to remove product on one hand and then to add more plant material. (Rickert,2007)
Can you imagine what a help the ancient archaea organisms can contribute to improve this existing manufacturing process? Using these heat resistant archaea as catalysts instead of the usual organisms, the fermentation reaction can then proceed at higher temperatures, up to 80 degrees centigrade, for example. At this temperature the archaea thrive best, the reaction rate will consequently be faster, and no expensive cooling step will be needed to activate the microorganisms.
This higher temperature also allows ethanol to be collected at the same time it is generated, by volatilization and subsequent distillation. There is no more need to stop the reaction to transfer the batch to another ethanol collecting vessel. And there is no need to stop to add more plant raw material; these can be added continuously into the reaction vessel. The downtime is therefore eliminated, as the process turns into a continuous conversion instead of batch fermentation.
Thus, it is desirable to do intensive research to test these theories, and establish new methodologies, which simplify the process of synthesis and increase recovery of product and which are capable of overcoming microbial sensitivity to product formation.( University of Nebraska 2007). Answering to the call and exploratory needs of the moment, several research institutions are now working on how to use archaea strains to make bioethanol production simpler, faster, less expensive, with higher yields and quality.
For example, at the Sandia National Laboratory in Livermore and UC Riverside scientists have made progress with their studies on archaea and plant decomposition. Researchers in this laboratory are occupied with work on Sulfolobus solfataricus strain of archaea which was first found in a dormant volcano near Naples and was discovered to have within it enzymes that could be useful in improving the current process of ethanol production. They found that the strain produces enzymes that swiftly break down the lignin in cell walls of plants, releasing the sugars within that can then be converted into ethanol. Now the scientists are working on improving the efficiency of the enzymes’ activity, increasing reaction speed and yield, in efforts to make fuel production more viable.
“We’re extremely excited about the future of this work,” Blake Simmons, leader of the
Sandia project, said Monday. “The ultimate dream — and it’s only a dream right now –
– would be to take a poplar tree, put it into a tank, let it sit for three days, and then
come back and watch as the ethanol comes pouring out of the spigot.”
( Perlman, 2007).
While this dream is still far from reality, the direction and impetus have been started to make the dream come true, in time.
In a laboratory at the University of Nebraska, another team of dedicated scientists have identified and patented a process using the same hyperthermophilic archaea strain Sulfolobus solfataricus. In their study, enzymes isolated from the archaea organism were glucose dehydrogenase and alcohol dehydrogenase. Ethanol was generated at high temperatures, specifically at its boiling point – thereby making its removal by volatilization faster. The quick removal of alcohol drives the reaction even more forward towards production of even more alcohol.
Discovery of this method makes possible the synthesis and recovery of product happening at the same time, in the same vessel, and overcomes problems which restrict current alcohol production efforts, such as enzyme inactivation by product accumulation and microbial ethanol sensitivity.
Jong-Shik Kim postdoctoral researcher and microbiology professor David Crowley both working at UC Riverside, recently made an important discovery. They found several hundreds of microbes still living in the La Brea pits. They were thriving without light and air in heavy oil and asphalt in soil which has been there for thousands of years.. Many were entirely newly discovered bacteia types, some were adapted to saline environment . Still others were highly resistant to radiation. There were also strains of Bacillus simplex extremophiles resistant to ultra violet radiation. (Perlman 2007).
In their amazing discovery they found that many of those microbes are also capable of breaking down cellulose and lignin in the same manner that archaea do, and that they also have similar enzymes that archaea have. As a result of this initial finding, his team is now using genetic engineering in an effort to cross transfer genes of these resistant bacteria to other bacteria species which can proliferate faster. Cultures of these combined genes bacteria will then be processed to harvest their enzymes which are the active ingredients which catalyze the fermentation of cellulose and lignin into ethanol.
Another Step in Biotechnology Advancement Featuring Archaea
Since strains of archaea were proven to be able to quicken decomposition of cellulose, and even lignin in wood plants, some companies have taken the exra step of incorporating the enzymes from archaea into corn that is grown for ethanol conversion. The Swiss company Syngenta, for instance, will soon be selling a genetically engineered corn which is designed to hasten its own degradation into ethanol. Each kernel of this self-processing corn contains the complex enzyme that must otherwise be added separately to the reactor vessels at the ethanol production facility (Tribe 2006).
Following the findings of the University of Nebraska, and the Sandia laboratories, Syngenta has produced a new version of the microbial enzyme amylase which becomes active when exposed to heat needed to prepare corn starch for ethanol production. The hybrid enzyme was generated from three closely related Archaea organisms – Thermococcales, Thermococcus, long familiar to microbiologists because their relatives make methane. The Archaea in this case comes from the bottom of the seafloor. This is very innovative and exciting modern biotechnology.
The catalyst works at temperatures above boiling water. To get corn to produce its own amylase, Syngenta inserted a gene borrowed from a type of archaea that live near hot-water vents on the floor of the ocean. The gene — actually a composite of three amylase genes — was developed with the help of Diversa, a San Diego company that specializes in finding chemicals in organisms that inhabit extreme environments. Not only that, Syngenta’s approaches cuts out manufacturing steps in amylase production by getting the corn plants to make their own catalyst amylase out in the corn fields. This is a truly an exceptionally brilliant green and clean approach( Tribe 2006).
It was announced that Syngenta‘s heat-resistant amylase starch liquification catalyst (AMY797E) will be incorporated in corn Line 3272 grain crops which will be used for dry grind fuel ethanol production in the United States (Tribe 2006). This development has raised some alarm from anti GMO activists who are afraid that this genetically engineered corn which is supposed to end up as biofuel might end up in the food chain. Therefore regulatory bodies are now carefully studying the permitting requiements for such innovation, and this new development will go through a rigorous regulatory procedure before being released or licensed for commercial consumption. Quite interesting to note that the ancient archaea, claimed to be the origin of life on earth can be party to a controversial GMO biotechnology concern.
Archaea have unique physical and biochemical properties that help it withstand varied harsh and extreme environments. Biofuels will have an increasing demand worldwide due to approaching concerns on the supply of fossil fuels and the increasing concerns on greenhouse gases causing global warming. Bioethanol in particular will be needed in increasing quantities as it is best qualified to partly replace transport’s energy needs. It was found that archaea strains can fulfill the need for a more efficient and less costly, but higher yielding production process to make bioethanol. The humble and ancient archaea which was on earth 3 billion years ago now holds enormous promise of assisting in the energy crisis by simplifying, speeding up and increasing recovery in production of biofuels, specifically ethanol using the fermentation process. Scientists have initially discovered the relevant mechanisms which have industrial applications and these have been tested in actual manufacturing operations set ups. Research laboratories are studying several archaea strains to discover and isolate more enzymes that have similar applications for ethanol production. They are using advanced techniques including modelling and high throughput screening methods to evaluate as many samples as possible. Genetic engineering has also been explored to create more cultures that carry the required enzymes but can multiply faster. As a further step, genes from a strain of archaea have even been inserted into a corn variety to hasten its later conversion to ethanol. This corn variant is for commercial release in 2008. In addition, other archaea strains, the methanogens, produce methane gas which can also be converted to ethanol using a process different from fermentation. Perhaps it is just a matter of time before this could also be explored by scientists as another pathway to produce biofuels.
Ahring, Birgitte.”Environment of Man: Biodiversity of Archaea” 2006.Biofuels:Production of Bioethanol. Environmental Microbiology and Biotechnology 2006
Antoni, Dominik,Vladimir V. Sverlov, Wolfgang H. Scharwz.“Biofuels From Microbes” 2007. Applied Microbial Biotechnology (2007)77:23-25.Springer-Verlag Sept 22 2007. http://www.springerlink.com/content/e2328823k467p181/fulltext.html
Perlman,David. Bacteria May Hold Promise in Making Biofuels” 2007.Energy Bulleti May 15 2007 http://energybulletin.net
Pollack,Andrew. “Redesigning Crops to Harvest Fuel”.2006. New York Times Sept 28 200 http://nytimes.com/2006/09/08/business/08crop.html
Rickert, Eve.”Ancient Microbes Could help Produce Biofuels”.2007.Black Gold Science and Technology. Mar 15 2007 http://www.aboutmyplanet.com
Priest, Winston, Series Ed. “The Evolution of Life”. 1994. Time Life Illustrated Encyclopedia of Science and Nature. Time Life Asia Edition.p33
Tribe, David, GMO Pundit. “Breakthrough Biotechnology Adds Value to Corn Growers Output to Reduce Cost of Ethanol Biofuel” June 4 2006
University of Nebraska research team.”Hyperthermophilic Enzymes for Indstrial Chemical Redox Reactions: A Method for Biofuel Ethanol Production” 2007.
Wright, Philip.”Proteonics Show Chemical Engineers the Answers” 2007