One way to increase world bio oil production that would cause a low ecosystem impact is to use lipids from single-cell oil microorganisms (also called oleaginous microorganisms), which present many significant advantages over plants. Oleaginous microorganisms, such as yeasts, fungi, bacteria, and microalgae, can accumulate high levels of lipids and do not require arable land, so in the conventional sense, they do not compete with food production.

Researchers in Spain at Universidad Rey Juan Carlos (URJC), have demonstrated the direct transformation of biomass consisting of the fungus M. circinelloides into biodiesel compliant with the U.S. ASTM D6751 and the EU’s EN14213 and 14214 standards. A paper on their work was published online back on April 2nd 2010 in the ACS journal Energy & Fuels.

Biodiesel consistsing of fatty acid methyl esters known as FAMEs has many advantages, such as high energy density, great lubricity, fast biodegradation rate, and reduced emissions of sulfur, aromatic compounds, and particulate matter. But biodiesel adoption is complicated because it competes with the food industry for the main raw material input, plant oils, and the worldwide supply of plant oils is limited by land and water availability.  There has been a rapid expansion in biodiesel production in not only developed countries most noticeably in the United States and the European Union, but also developing countries are working to substitute fuel production for subsistence farming.

Oleaginous yeasts and fungi have also been considered as potential oil sources for biodiesel production because they accumulate large amounts of lipids. Among these microorganisms, particular attention has been dedicated to various oleaginous zygomycetes species, such as Mortierella isabelina and Cunninghamella echinulata, which may accumulate up to 86% and 57% of lipids in the dry biomass, respectively.

Now for the plus or minus depending on your point of view, The experimental fungi are able to grow and accumulate large amounts of lipids in cultures containing raw glycerol sourced from biodiesel production as a carbon source. Glycerol is the major byproduct of the biodiesel production, and its recycling to produce oleaginous microbial biomass could significantly increase the unit revenue of biodiesel production.

The results of the lab production are very encouraging.  The URJC team cultivated M. circinelloides in a liquid medium containing glucose as a carbon source at 20 grams per liter. Under the experimental conditions, the fungus grew very quickly consuming all of the available glucose and stopped growing in the first 48 hours after inoculation the spores into the liquid medium. After 96 hours, the team obtained 4.17 ± 0.25 g/L fungal biomass with a total lipid content of 22.9 ± 0.9% dry mass.

Fungal Biomass Biodiesel Production Diagram. Click image for the largest view. Image Credit: Universidad Rey Juan Carlos

The saponifiable lipids, those that can be transformed into the desired FAMEs and free fatty acids (including energy storage and structural lipids) were 98.0 ± 1.3% of the total lipids extracted from the biomass.  This is rich stuff.

With the experiment’s production at such high concentration of the free fatty acids the team determined that an acid-catalyzed process was more suitable for producing biodiesel than an alkali one to avoid yield losses from free fatty acid neutralization.

The team used the acid-catalyzed direct transformation method with methanol and chloroform as solvents; with trials of  H2SO4, HCl, and BF3 as acid catalysts. The team found optimal reaction conditions at 8 hours at 65 °C. The respective biodiesel yields were 18.9, 18.9, and 18.4% relative to the dry mass using the three catalysts.

The fungus strain is called MU241 derived from R7B after replacement of its leuA mutant allele by a wild-type allele, was used as a wild-type strain to produce fungal biomass.  The team reports, “The level of neutral lipids (storage lipids) increased with time during the cultivation of this fungus, which means a decrease in the relative proportion of all of the structural lipids with this variable. In fact, the amount of structural lipids in a microorganism is concrete, and therefore, it has to keep constant with time. In contrast, lipid accumulation in M. circinelloides was 18.9% at 24 hours, increasing only slightly after this time.”

The team compared production of their MU241 strain working in a liquid with known solid growth results.  There results suggest that the fungal biomass from liquid cultures in the dark shows better characteristics for biodiesel production than that from solid cultures.

Not only is the product rich, its very high quality.  Depending upon the catalyst used, the ester content ranged between 99.0 and 99.2%, which is significantly higher than the corresponding specified minimum value in the European Union standard of 96.5%. These values were higher and the reaction was faster than those reported for other oleaginous microorganisms, in which an acid-catalyzed direct transformation method was used. Furthermore, the amounts of all the byproducts analyzed were below the maximum allowed values for American and European standards. Thus, the contents of individual glycerides (mono-, di-, and triglycerides) were within the biodiesel specifications, indicating that the transesterification and esterification reactions were complete.

The bio diesel is also quite chemically clean – the free glycerol was lower than the two standard limits showing the glycerol residues were eliminated during the purification treatment making the individual glyceride and free glycerol levels below the established limits. The total glycerol content also met all of the standards.  The acid values were also within the specifications in all reactions. Additionally the non-saponifiable lipids were not detected in the new biodiesel, which means that these types of lipids were also eliminated during the purification stage.

There remains but one small problem – the biodiesel obtained had small quantities of polar lipids, which were lower than 0.9% in all cases. These compounds are residuals of nonconverted polar lipids, and they are not as of yet considered in the established biodiesel specifications.

The Spanish team can be quite proud of their achievement; efficient biodiesel production by direct transformation of fungal biomass without lipid extraction is technically feasible, which represents a starting point for developing this process on an industrial scale.

Fungal research might need to catch up with bacterial and algal efforts in the genetic manipulation area.  Questions remain on feeding the fungus – coming up with glycerol isn’t a major answer – especially as the U.S. Congress has lunched, dined and partied its way out of keeping the biodiesel seed incentives in place and the industry healthy enough for progress.  The bio middle distillate market is in deep trouble in the U.S. with only the military and airframe manufacturers still providing support.  It’s a warning shot, well artillery, shot warning to any industry that the economy needs to develop new energy and fuel resources.

The U.S. is only 25% of the world market leaving a huge opportunity out there. The Spanish research goes far to getting a new path on the table, intensifying the competition for bio diesel   It’s a pity the U.S. is essentially out, there are great resources that could put strong legs under the Spanish achievement’s potential.  But, it will come – the results are just too good to overlook.


3 Comments so far

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  2. Libby on October 20, 2010 8:58 AM


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