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Direct Production of Biodiesel from the Fungus M. circinelloides; Opportunity to Enhance Yield with Genetic Engineering

Researchers in Spain have demonstrated the direct transformation of biomass consisting of the fungus M. circinelloides into biodiesel compliant with ASTM D6751 and EN14213 and 14214 standards. A paper on their work was published online 2 April in the ACS journal Energy & Fuels.

Oils from oleaginous microorganisms, such as yeasts, fungi, bacteria, and microalgae, are under investigation as alternatives to plant—and especially food crop—oils as feedstocks for renewable fuels and chemicals. Algae are especially of interest because of their ability to capture CO2 in lipids, but cost-effective, large scale production is still problematic, note Vicente et al. in their paper. Furthermore, not all oleaginous microorganisms have ideal lipid profiles for biodiesel production.

On the other hand, lipid profiles could be modified by genetic engineering in some oleaginous microorganisms, such as the fungus Mucor circinelloides, which has powerful genetic tools. We show here that the biomass from submerged cultures of the oleaginous fungus M. circinelloides can be used to produce biodiesel by acid-catalyzed direct transformation, without previous extraction of the lipids. Direct transformation, which should mean a cost savings for biodiesel production, increased lipid extraction and demonstrated that structural lipids, in addition to energy storage lipids, can be transformed into FAMEs.

—Vicente et al.

The team grew M. circinelloides (strain MU241) in a liquid medium containing glucose as a carbon source (20 g/L). Under experimental conditions, the fungus grew very quickly because it consumed all of the available glucose and stopped growing in the first 48 hours after inoculation. After 96 h, they obtained 4.17 ± 0.25 g/L fungal biomass with a total lipid content of 22.9 ± 0.9% dry mass.

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

Because of the high concentration of free fatty acids (3.6 ± 0.6%) in M. circinelloides , 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.

They used the acid-catalyzed direct transformation method with methanol and chloroform as solvents and H2SO4, HCl, and BF3 as acid catalysts. Using optimal reaction conditions (8 h at 65 °C), biodiesel yields were 18.9, 18.9, and 18.4% relative to the dry mass of M. circinelloides, using H2SO4, HCl, and BF3, respectively.

These yields were even slightly higher than the corresponding theoretical yield calculated for this microorganism (18.1%), indicating that acid-catalyzed direct transesterification/esterification of fungal biomass can be applied to M. circinelloides biomass from submerged cultures because it improves the amount of total lipids extracted in comparison to the conventional methods for lipid extraction from microorganisms.

This observation is supported by previous works describing increased recovery of fatty acids from microorganisms by direct transesterification techniques. Interestingly, these results also indicate that saponifiable lipids other than triglycerides, such as phospholipids, sphingolipids, and saccharolipids, are transformed into FAMEs by this method and should be considered as substrates for FAME obtention.

...biodiesel yields should be increased to make the industrial process economical, which could be attained by the genetic manipulation of this fungus. In this sense, efforts are now dedicated to overexpress genes that code for enzymes postulated to be rate-limiting steps for fatty acid biosynthesis in oleaginous fungi. Other strategies are focused on the generation of strains with enhanced ability to use crop residues or industrial byproduct, avoiding competition with the food supply, with low linolenic acid levels or overexpressing genes involved in saponifiable lipid biosynthesis.

Particularly interesting is the generation of strains with low free fatty acid levels because they could be used for biodiesel production by using a base-catalyzed technology, which is the common way to produce biodiesel on an industrial scale.

—Vicente et al.


  • Gemma Vicente et al. (2010) Direct Transformation of Fungal Biomass from Submerged Cultures into Biodiesel. Energy Fuels, Article ASAP doi: 10.1021/ef9015872



The yield of fungal biomass from sugars is ~23%, and the yield of FAME from that biomass is ~19% at best.  Net yield, about 4.4%.

This doesn't look like a process with a future.

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