Biodiesel production from biomass of an oleaginous fungus

https://doi.org/10.1016/j.bej.2009.07.014Get rights and content

Abstract

The present paper introduces the filamentous fungus Mucor circinelloides as a potential feedstock for biodiesel production. These microbial lipids showed a high content (>85%) of saponifiable matter and a suitable fatty acid profile for biodiesel production. The effectiveness of the lipid extraction process was studied for three different solvent systems: chloroform:methanol, chloroform:methanol:water and n-hexane. Biodiesel was produced by acid-catalysed transesterification/esterification following two different approaches: transformation of extracted microbial lipids and direct transformation of dry microbial biomass. After 8 h of reaction at 65 °C in the presence of BF3, H2SO4 or HCl as acid catalysts, the direct process produced fatty acid methyl esters (FAMEs) with higher purities (>99% for all catalysts) than those from the two-step process (91.4–98.0%). In addition, the yield was also significantly higher in the direct transformation due to a more efficient lipid extraction when the acid catalyst was present.

Introduction

Demand for fatty acid methyl esters (FAMEs) as diesel fuel (biodiesel) has increased significantly due to the instability of petroleum prices and the development of government measures in many countries around the world that establish a minimum proportion of biofuel for all petrol and diesel used in transport. For instance, the European Union establishes a minimum content of 5.75% of biofuel by 2010 (European Union Directive 2003/30/EC) and the United States plans to increase the amount of bioethanol and biodiesel to 12.95 and 36 billion gallons by 2010 and 2022, respectively (Energy Independence and Security Act of 2007).

Biodiesel constitutes a renewable fuel that is compatible with current commercial diesel engines and has clear benefits relative to diesel fuel including enhanced biodegradation, reduced toxicity and a lower emission profile [1]. Nonetheless, biodiesel presents some disadvantages. One of its drawbacks is the high manufacturing cost, which is mainly due to the high cost of the vegetable oil. Actually, 70–90% of the biodiesel production cost corresponds to raw vegetable oil. In addition, the biodiesel industry competes with the food industry for oil crops. In fact, it has been calculated that a very large percentage of the current available arable land is required to achieve the current biofuel objectives using crops such as rapeseed or sunflower. Therefore, it is necessary to explore new raw materials that reduce the biodiesel price without competing with food production. In this context, oils from microorganisms (also called single-cell oils) constitute a promising alternative for producing biodiesel since they present many advantages over vegetable oils from oleaginous plants. Microorganisms can accumulate high level of lipids and do not require arable land. In addition, the production of these microorganisms does not compete with food production since biomass residuals can be used as carbon source.

Microorganisms which accumulated more than 20–25% lipids are usually referred to as oleaginous species [2]. In most cases, the oil from these microorganisms is in the form of triglycerides, which are also the main component in vegetable oils and animal fats. Therefore, the microbial lipids can potentially be used as raw material for biodiesel production using the common way to produce FAMEs in the biodiesel industry, i.e. the transesterification reaction with methanol in the presence of a basic catalyst (e.g. sodium and potassium hydroxide or sodium methoxide). However, the utilisation of these catalysts in the transesterification of vegetable oils or animal fats with a high concentration of free fatty acids produces soaps by neutralisation, which in turn partially consumes the catalyst, decreases the biodiesel yield and complicates the separation and purification steps [1]. Free fatty acids are usually present in the lipid composition of microbial cells [3]. However, soap formation from free fatty acid neutralisation can be avoided by using an acid catalyst such as sulphuric or hydrochloric acids. The acids catalyse the free fatty acid esterification with methanol to also produce FAMEs, increasing the biodiesel yield [4].

The principal oleaginous microbial species are microalgae, bacteria, fungi and yeasts. The use of microorganisms as a source of lipids has been extensively investigated for their application as food additives, pharmaceuticals and feed ingredients for aquaculture [2], [3], [5], [6], [7], [8], [9], [10]. Microorganisms are sources of edible oils because they have the ability to produce oils rich in polyunsaturated fatty acids, which are in demand as dietary supplements and for infant nutrition [7]. More recently, some works have dealt with the use of oleaginous microorganisms for biodiesel production. Particularly, microalgae, which capture carbon dioxide by transformation into lipids using sunlight, have attracted recent attention and investment for biofuel production because of their higher oil productivity and faster growth compared to conventional energy crops [11], [12]. However, these photosynthetic microorganisms have problems associated with their growth in bioreactor systems due to the necessity of light supply and large acreages. In addition, the economics of producing biodiesel from microalgae need to improve to make it competitive with diesel [11]. Through changing culture conditions or using genetic engineering modifications, some autotrophic microalgae can be converted to heterotrophic microalgae and such heterotrophic microalgae can also accumulate oils using organic carbon as the carbon source instead of CO2 [13]. According to Miao and Wu [14], heterotrophic growth of a microalga (Chlorella protothecoides) results in higher biomass production and higher lipid accumulation in cells in comparison to the autotrophic growth of this microalga. These authors reported an integrated method for biodiesel production from heterotrophic C. protothecoides oil by acidic transesterification. Conversely, little information has been reported so far on the use of lipids from yeast, fungi and bacteria for biodiesel production. Nonetheless, two recent reviews dealt with the related research about these oleaginous microorganisms, and the prospects of such microbial oils for biodiesel production [13], [15]. In comparison to the microalgae, the growth of these microorganisms can be carried out in conventional microbial bioreactors, which will improve the biomass yield and will reduce the cost of biomass and oil productions. In this context, one work described the acid methanolysis of biomass from two yeasts (Lipomyces starkeyi and Rhodosporidium toruloides) and one filamentous fungus (Mortirella isabellina) for biodiesel production [16].

In the present work, we have investigated the production of biodiesel from the fungal Mucor circinelloides. This fungus shows many relevant features favouring its use for biodiesel production, including a high level of lipids in the mycelium (around 25% dry mass in wild-type strains) [7], good biomass production during submerged batch cultivation in bioreactors using a wide range of carbon sources [17], and a proven capacity to grow in large industrial stirred-tank fermenters (220 m3) to produce oil rich in γ-linolenic acid [10]. More significantly, the regulation of lipid accumulation in this fungus has been extensively studied [18], and key genes have been identified that could be manipulated using a large number of already available molecular tools, including gene silencing (RNAi) [19]. Based on its potential use in biofuel production, the Department of Energy of the United States (DOE) has selected this fungus to sequence its genome through the bioenergy program at the Joint Genome Institute, a project that is nearly complete.

The present study included the lipid extraction and characterisation, in addition to the biodiesel production from M. circinelloides biomass. Two procedures for biodiesel production were compared: the extraction of lipids from M. circinelloides biomass followed by the transformation of the extracted lipids into FAMEs and the direct conversion of the M. circinelloides biomass, without previous extraction, to produce FAMEs. The high quality of the biodiesel produced using the direct method, which complies with the European and American standards, suggests that M. circinelloides biomass could be used as a feedstock to produce biodiesel.

Section snippets

Strain and growth conditions

The strain MU241, derived from R7B [20] after replacement of its leuA mutant allele by a wild-type allele, was used as a wild-type strain to produce fungal biomass. For biomass production, 2.5 × 105 spores per plate (9.5 cm diameter) were inoculated on solid minimal medium pH 4.5 (YNB; [21]) with a cellophane sheet and incubated for three days at 26 °C in the presence of white light (4.8 W m−2). Mycelia grown on the cellophane sheet were harvested and dried between paper towels, frozen in liquid

Microbial biomass production and lipid extraction

To produce biodiesel, M. circinelloides biomass was obtained from the prototrophic strain MU241 grown on a solid minimal medium containing glucose as a carbon source (10 g l−1). After three days of growth, a 3.73 ± 0.27 g l−1 of fungal dry mass was obtained. Lipids were extracted from this fungal biomass using three mixtures of solvents: chloroform:methanol (C:M), chloroform:methanol:water (C:M:W) and n-hexane. Table 1 shows the lipid content, the biomass free of lipids and, therefore, the extraction

Conclusions

The present work shows that lipids from M. circinelloides may be a suitable feedstock for biodiesel production. The chloroform:methanol extraction system showed the highest lipid yield among the three extraction processes studied. FAMEs were produced by acid-catalysed transesterification and esterification at 65 °C for 8 h comparing two different approaches: the transformation of previously extracted lipids and direct transformation from fungal biomass. This study shows that the direct method not

Acknowledgements

This work was funded by the D.G. de Investigación y Política Científica (Comunidad Autónoma de la Región de Murcia, Spain), project BIO-BMC 07/01-0005.

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