Conversion of cardoon crop residues into single cell oils by Lipomyces tetrasporus and Cutaneotrichosporon curvatus: process optimizations to overcome the microbial inhibition of lignocellulosic hydrolysates

Highlights

• Efficient conversion of cardoon residues into single cell oils through L. tetrasporus.

• C. curvatus was not able to grow in undetoxified cardoon hydrolysate.

• Lipids yields of 20% and lipid cell content of 47% were achieved.

• The production of mannitol from L. tetrasporus was reported for the first time.

• Fed batch process in bioreactor produced 12.7 g L^-1 lipids with high oleic acid (59%).

Abstract

In the present work, the production of Single Cell Oils (SCOs) from undetoxified cardoon hydrolysates (UCH) was investigated. The raw material was pretreated by acid catalyzed steam explosion at 195 °C for 7.5 min and hydrolysed at high dry matter content to achieve a final hydrolysate containing 90 g L^-1 glucose and 9 g L^-1 xylose. The different ability of Cutaneotrichosporon curvatus and Lipomyces tetrasporus to overcome the toxic effect of the biomass degradation by-products, mainly furan derivatives, in different growth phases was investigated in different set-ups conditions including four hydrolysates concentrations, (glucose and xylose respectively 30 + 3, 45 + 4, 60 + 6, and 90 + 9 g L^-1) and two fermentation modes, batch and fed batch. The results indicated that the inoculum age corresponded to a different metabolism of the furanic aldehydes only in L. tetrasporus. C. curvatus was severely inhibited by UCH in any growth phase. L. tetrasporus cells in the stationary phase (SP) grew in UCH at any concentration, while cells in the exponential phase (EP) grew only at the highest dilution rate. The maximum lipids yields and lipid cell content of 20% and 47% respectively (7.1 g L^-1 lipids) were achieved in batch mode using SP-inocula and the highest dilution ratio. The dominant fatty acid was oleic acid (59%). The scale-up to the 2 L bioreactor, in fed-batch mode, increased the lipids concentration up to 12.7 g L^-1 and the productivity up to 0.135 g L^-1 h^-1.

Introduction

Nowadays, the most dominant resources for world energy supply derive from crude oil, coal and gas. The limited reserve of such resources and the environmental concerns due to the increased deployment in the last years has stimulated the development of novel processes based on the use of renewables. Biorefineries represent novel productive models aimed at converting renewable feedstocks and their intermediates products, platforms molecules, to achieve fuels, chemicals, materials and power by using cascading approaches. In Italy, the Matrica Biorefinery in Porto Torres is developing a cardoon-based local value chain to produce and convert oils to many biobased products. Oils are a valuable platform for a number of industrial applications including the production of oils-based biofuels, hydrocarbons and several chemical intermediates for green chemistry applications. Besides the production of biodiesel, monounsaturated fatty acids can be transformed via ozonolysis into dicarboxylic acids, important intermediates for polyesters and polyamide synthesis (Kadhum et al., 2012). Furthermore, fatty alcohols, synthetized by fatty acid hydrogenation, find their application in formulations of detergents, surfactants, cosmetics, pharmaceuticals, etc. (Sánchez et al., 2017). Epoxidation of oleic acid, could be used for the synthesis of polyols, glycols, plasticizers for PVC compounds and polymers used like drug delivery vehicles (Jin et al., 2015).

In nature, many oleaginous yeasts are able to accumulate SCOs in the form of lipids, primarily as triglycerides (TAGs) and fatty acids (FAs) up to 70% of their Dry Cell Weight (DCW). The industrial production of yeast SCOs is still in its infancy but have attracted much attention due to the close similarity of the FAs profile to that of oils from agricultural oil crops, which make them generally suitable for processing into several final products (Probst et al. 2016). As an example, the transesterification of TAG produced Fatty Acids Methyl Esters (FAME) that meet the quality criteria for the ASTM D6751-12 biodiesel standard. (Saenge et al. 2011). The most investigated yeasts species are Yarrowia lipolytica, Rhodotorula glutinis, Lipomyces starkeyi, Cutaneotrichosporon curvatus, and Rhodosporidium toruloides. These yeasts, can grow on several carbon sources including many C6 and C5 sugars. The life cycle of these microorganisms is characterized by three physiological phases: balanced growth phase, oleaginous phase and lipid turnover phase (Dourou et al., 2018). In the growth phase, sugars are converted into microbial biomass. In this phase the microorganism accumulates ATP and reducing equivalents such as NADH or NADPH useful for the biosynthesis of various macromolecules. The oleaginous phase is triggered by carbon excess and absence of nutrients, usually nitrogen but also phosphorus, magnesium, zinc and iron (Beopoulos et al. 2009). The nitrogen starvation leads to a decrease of intracellular AMP, used as NH₄⁺ source resulting in the inhibition of the mitochondrial isocitrate dehydrogenase, (ICDH) (Ratledge and Wynn 2002). Inhibition of ICDH marks the beginning of the lipogenesis phase (Papanikolaou et al., 2004). In addition to ICIDH, fundamental enzymes in the lipids synthesis are: ATP-citrate lyase which cleaved citric acid into acetyl-coA and oxaloacetate and the malic enzyme which converts malate into pyruvate with the production of reducing equivalents (NADP and NADPH), essential for the synthesis of FAs. When the extracellular carbon runs out, the lipid turnover phase is triggered and the lipids storage is used as an energy source as long as essential nutrients are present in the medium.

A recent techno-economic analysis, determined that one main limitation to the process feasibility is the cost of the glucose based media affecting the overall oils production cost by 38% (Koutinas et al., 2014). The use of agricultural by-products could be an option to produce low cost carbon sources. In fact, residual lignocellulosic feedstocks have the potential to be used as raw materials to produce sugars.

A pre-treatment step is required to disrupt the biomass structure (Di Fidio et al., 2020). Depending on the process severity, side stream products can be generated due to the degradation of carbohydrates and lignin. These compounds could inhibit the yeast growth and the final SCO yields (Sitepu et al., 2014). Generally, the microbial inhibition due to degradation by-products varies from one microorganism to another and depends on the composition of the fermentation medium (Wierckx et al., 2011). Cells typically detoxify the media by reducing the organic aldehydes, 5-Hydroxymethylfurfural (5-HMF) and furfural (2-Fur), to less toxic products, namely furan-2,5-dimethanol (FDM) and furfuryl alcohol (Fur-OH) (Flores-Cosio et al., 2018; Liu et al., 2008).

In most cases the toxic effect of furanic aldehydes is due to their ability to denature the cytoplasmic proteins, to bind and alter the composition of the membrane lipids thus disturbing the metabolites transport (Favaro et al., 2016). Furthermore they often act as direct competitors in many cellular metabolic cycles interfering with glycolytic enzymes, like hexokinase and glyceraldehyde-3-phosphate dehydrogenase (Almeida et al., 2007). The reduction of aldehydes to alcohols is carried out by enzymes belonging to the family of alcohol dehydrogenases making use of NADH or NADPH as a cofactor (Liu et al., 2008). Since NADH plays a fundamental role in glycolytic cycle, the shortening of NADH impedes the glucose catabolism. This stress typically delays and sometimes damages the metabolic processes often lengthening the latency phase (Flores-Cosio et al. 2018).

Thus, to increase the process yields, it is important to increase the yeasts tolerance to inhibitors in biomass hydrolysates (Wang et al., 2018). In general, to do this, specific strain-dependent adaptation protocols were developed aimed at acclimating the yeast cells to the current inhibitors level (Auesukaree, 2017; Saini et al., 2018; Lewis et al., 1993). More interestingly, it is known that many yeasts are able to activate different mechanisms, both specific and general, to increase the stress tolerance especially in the stationary phase of growth. For instance, the SP-cells of Saccharomyces cerevisiae yeast demonstrated an increased cell robustness to heat shock, osmotic stress, free-thaw stress and weak acids stress (Werner‐Washburne et al., 1996). The higher robustness of SP-cells is often due to the activation of multiple cellular regulatory events upon nutrient starvation, including the environmental stress response, which leads to the adaptation of cellular resources to ensure survival in difficult environments (Gasch, 2003; Smets et al., 2010). On the whole, while the metabolism of the main inhibiting products was studied extensively on the performance of ethanol-producing microorganisms, less research has been dedicated to oleaginous yeasts.

In the present paper, lignocellulosic biomass were obtained from cardoon crops harvested in the Matrica biorefineries (Porto Torres, Italy), and converted to SCOs by using Cutaneotrichosporon curvatus and Lipomyces tetrasporus. The aim of the study was to find the optimal process conditions for converting undetoxified biomass hydrolysates, into TAGs, by optimizing several parameters. In particular, cells harvested from two growth phases, SP and EP, were tested at different process conditions and used as process strategy to enhance the yeast resistance to microbial inhibitors. To the best of our knowledge this approach has never been tested before for oleaginous yeasts.