Biotechnological conversions of bio-diesel derived waste glycerol into added-value compounds by higher fungi: production of biomass, single cell oil and oxalic acid

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Abstract

Waste bio-diesel derived glycerol was used as the sole carbon source by higher fungi; two Lentinula edodes strains were flask cultured in carbon-limited conditions and displayed satisfactory growth in media presenting weak agitation, pH 4.0 and temperature 25 °C. Maximum biomass of 5.2 g/l was produced. Mycelia were synthesized, containing around 0.1 g of fat per g of biomass, with linoleic acid (Δ9,12C18:2) being the principal cellular fatty acid produced. Two Aspergillus niger strains were grown in nitrogen-limited flask cultures with constant nitrogen and two different initial glycerol concentrations into the medium. In 250-ml flask cultures, large-sized pellets were developed, in contrast with the trials performed in 2-l flasks. Nitrogen limitation led to oxalic acid secretion and intra-cellular lipid accumulation; in any case, sequential production of lipid and oxalic acid was observed. Initially, nitrogen limitation led to lipid accumulation. Thereafter, accumulated lipid was re-consumed and oxalic acid, in significant quantities, was secreted into the medium. In large-sized pellets, higher quantities of intra-cellular total lipid and lower quantities of oxalic acid were produced and vice versa. Maximum quantities of oxalic acid up to 20.5–21.5 g/l and lipid up to 3.1–3.5 g/l (corresponding to 0.41–0.57 g of fat per g of biomass) were produced. Lipid was mainly composed of oleic (Δ9C18:1) and linoleic (Δ9,12C18:2) acids.

Introduction

Bio-diesel fuels, defined as principally methyl- and to lesser extent ethyl- or butyl-esters deriving from trans-esterification of low value vegetable or animal fats, already constitute an alternative type of fuel for various types of diesel engines and heating systems (Johnson and Taconi, 2007, Papanikolaou, 2008). Due to the potential exhaustion of conventional fuels and the various environmental issues imposed, the application of bio-fuels in a large commercial scale is strongly recommended (e.g. EU directive 2003/30/EC which plans to introduce 5.75%, w/w, of bio-fuel in the conventional fuel by 2010). This fact may have as result the accumulation of tremendous quantities of glycerol into the market in the near future (Johnson and Taconi, 2007). In 2007 only in Europe, an over-capacity of more than 6 × 105 metric tones of glycerol occurred (Papanikolaou, 2008, Papanikolaou, 2009); to give the magnitude of the imposed problem, it is pointed out that with the production of 10 kg of bio-diesel deriving from trans-esterification of various oils, 1 kg of (pure) glycerol is generated (Johnson and Taconi, 2007, Papanikolaou, 2008). Therefore, glycerol over-production and disposal is likely to cause serious environmental problems in the near future (in Germany in 2007, crude glycerol deriving from various bio-diesel plants was already treated as a typical “industrial waste-water”—Papanikolaou, 2008); conversion, thus, of this low- or, even, negative value material to higher added-value products by the means of chemical or fermentation technology currently attracts much interest. Thermo-chemical processes have already appeared in the international literature that deal with conversion of glycerol into propylene glycol, acetol, hydrogen or various other compounds (Cortright et al., 2002, Dasari et al., 2005, Chiu et al., 2006, Alhanash et al., 2008). The biotechnological (fermentative) valorization glycerol has also been substantially developed during the last years, and various microbial added-value metabolites (e.g. 1,3-propanediol, organic acids, storage lipids, carotenoids, butanol) have been produced through utilization of this waste stream as microbial substrate (Morgunov et al., 2004a, Johnson and Taconi, 2007, Koutinas et al., 2007, Koutinas et al., 2008, Mu et al., 2008, Papanikolaou, 2008, Papanikolaou, 2009, Xiu and Zeng, 2008, Mantzouridou, 2009, Papanikolaou and Aggelis, 2009, Taconi et al., 2009, Wen et al., 2009a, Wen et al., 2009b).

Aim of the current investigation was to demonstrate potential biotechnological ways of valorizing crude glycerol stemming from bio-diesel industries, by using this renewable substrate as carbon source of natural fungal species. Specifically, the present study was focused upon the transformation of crude glycerol into organic acids (principally oxalic acid) and microbial lipid (the so-called “single cell oil—SCO”) by using Aspergillus niger strains and microbial mass by using Lentinula edodes strains. A. niger is an ascomycetous mold very often used in food technology and white biotechnology; strains of the above-mentioned microorganism have been successfully employed in bio-processes related with the production of organic acids (such as citric acid and to lesser extent gluconic acid) or enzymes (Soccol et al., 2006, Papagianni, 2007). However, to the best of our knowledge, with an exception of a recent book chapter (Musiał and Rymowicz, 2009) (crude) glycerol has never been used as substrate by strains of this microorganism. Although in some manuscripts production of oxalic acid by Aspergillus strains has been reported (Soccol et al., 2006, Papagianni, 2007), in most of the cases carbon substrates other than glycerol (e.g. glucose, crude fatty acids, whey) have been used for this purpose. As far as the microorganism L. edodes is concerned, it is a basidomycetous mold known also as Shiitake; this fungus is a medicinally important edible mushroom (Israilides and Philippoussis, 2003, Philippoussis et al., 2007). Only the last years some research has been made using L. edodes as a potential biotechnological tool, with studies focused upon the production of anti-tumor and anti-bacterial agents (Hassegawa et al., 2005, Surenjav et al., 2006, Israilides et al., 2008) or enzymes (Cavallazzi et al., 2005). In some cases, due to the capability of strains of this species to produce extra-cellular oxidizing enzymes (e.g. laccases) this microorganism has been successfully used in the simultaneous detoxification (removal of phenolic compounds and color) of phenol-rich residues (i.e. olive-mill waste-waters) and the production of enzymes and biomass (D’Annibale et al., 2004). In any case though, glycerol has never been used as substrate by strains of this mushroom.

As previously stated, while oxalic acid has rarely been produced through microbial conversion of glycerol (Musiał and Rymowicz, 2009), this substrate has been used as carbon source by a number of eukaryotic microorganisms (principally wild or mutant Yarrowia lipolytica strains) and citric acid (Papanikolaou et al., 2002, Papanikolaou et al., 2008, Rymowicz et al., 2006, Rymowicz et al., 2008, Imandi et al., 2007, Papanikolaou and Aggelis, 2009, Makri et al., 2010) or puryvic acid (Morgunov et al., 2004a) has been produced. On the other hand, conversion of (waste) glycerol into SCO has been investigated in a number of works, in which oleaginous fungi (Mantzouridou et al., 2008, Papanikolaou et al., 2008, Fakas et al., 2009a, Fakas et al., 2009b, Wen et al., 2009b), (heterotrophically growing) algae (Chi et al., 2007, Pyle et al., 2008, Wen et al., 2009b) or yeasts (Meesters et al., 1996, Papanikolaou and Aggelis, 2002, Papanikolaou and Aggelis, 2009, Makri et al., 2010) have been used. Finally, glycerol has been utilized as substrate by eukaryotic microbial strains in order for biomass to be produced (Kim et al., 2000). The current investigation aimed at assessing potentialities of bio-valorization of waste glycerol by using L. edodes and A. niger strains in order for the production of microbial mass, organic acids and SCO to be performed. Biochemical and kinetic considerations related with the growth of these microorganisms on glycerol were considered and discussed.

Section snippets

Microorganisms and media

A. niger strain NRRL 364 was kindly provided by the NRRL culture collection (Peoria, USA). Another newly isolated A. niger strain was also used in the present study. This strain was isolated from edible products (fat-rich foods), was identified and characterized in the Laboratory of Food Microbiology and Biotechnology (Department of Food Science and Technology, Agricultural University of Athens, Greece). The culture number given to this newly isolated A. niger strain was LFMB 1. L. edodes

Cultures of L. edodes on crude glycerol

L. edodes strains AMRL 119 and AMRL 121 were flask cultured in carbon-limited conditions in order to direct microbial metabolism towards the synthesis of biomass. In a first approach, cultures were performed at initial pH 5.0, T = 28 °C and agitation of 180, 60 and 0 rpm in order to evaluate the impact of the agitation speed upon the microbial growth. In general, very limited growth was observed after 12 days, especially for L. edodes cultures carried out at the high speed (180 rpm); in the

Discussion

In the present study, submerged flask cultures of L. edodes strains were accompanied by relatively small biomass production when crude glycerol was used as the sole substrate in carbon-limited experiments. Mycelia were able to grow at 60 rpm but not at 180 rpm, suggesting significant strain sensitivity against the shearing forces caused by a powerful agitation. The initial pH of the culture medium and the incubation temperature seemed to have an important impact upon the production of biomass by

Acknowledgement

The present work was financed by the ΠABET-2005 project (05ΠAB105) entitled “Valorization of agro-industrial residues through the cultivation of the mushroom fungus L. edodes for the production of metabolic products of biotechnological interest” (General Secretariat of Research and Development, Greek Ministry of Development).

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