Fermentation Performance of Oleaginous Yeasts on Eucommia ulmoides Oliver Hydrolysate: Impacts of the Mixed Strains Fermentation

Eucommia ulmoides gum (EUG), a trans-1, 4-polyisoprene natural polymer, is an important natural biomass rubber that can be used as elastic materials, thermoplastic materials, shape-memory materials, etc (Kang et al., 2018, Sun et al., 2018, Xia et al., 2019). EUG is usually extracted from the leaves, bark and samara of Eucommia ulmoides Oliver (EUO), a widely distributed valuable economic plant in China (Yang et al., 2020). Different from natural rubber which is composed of cis-1, 4-polyisoprene and can be gathered from the sap of plants, the extraction of EUG from EUO mainly includes three steps: pretreatment, extraction and purification, and pretreatment is considered the most important step that can efficiently destroy the EUG-containing cell wall and improve the yield of EUG (Qin et al.,2021). Compared with the mechanical process (Wei et al., 2019) or alkaline pretreatment and fermentation (Liu et al., 2010, Zhang et al., 2009), dilute acid hydrolysis pretreatment is gaining more and more applications because of its high efficiency and EUG yield (Qin et al., 2021, Yang et al., 2020). More importantly, dilute acid hydrolysis is a state-of-the-art chemical technology for the conversion of lignocellulosic biomass to fermentable sugars and other bio-based platform compounds (Besson et al., 2014, Huang et al., 2019, Xu et al., 2016). Thus, the wastewater produced in the pretreatment process, namely Eucommia ulmoides Oliver hydrolysate (EUOH), can be treated and re-utilized through microbial treatment technology, so as to further increase the added value of the EUG production and reduce environmental pollution.

As a sort of lignocellulose biomass, EUO contains a large amount of cellulose and hemicellulose, making the EUOH contain a variety of carbohydrates, mainly including glucose, xylose, arabinose, cellobiose, etc., as well as a small amount of volatile fatty acids (VFAs) dominated by acetic acid (Yang et al. 2020). And when organic acids (e.g. formic acid, acetic acid) are used as catalyst in the dilute acid hydrolysis process, the possible residual acids will further increase the total organic acids concentration in the EUOH. It is well known that xylose and arabinose (five-carbon sugars) usually cannot be fermented by most yeasts including native Saccharomyces cerevisiae strains (Cai et al., 2012). In particular, xylose, as the final hydrolysate of hemicellulose, is the most abundant pentose sugar in EUOH as well as in nature. However, the current utilization of xylose is limited to the production of feed yeast, furfural, and xylitol (Finneran and Popovic, 2018, Ghindea et al., 2010, Zhao et al., 2020). How to expand and optimize the utilization of pentoses represented by xylose is of great significance to fermentation engineering. In addition, it is reported that organic acids, which also contained in EUOH, could have certain inhibitions on the growth of microorganisms. Especially, the high concentration of acids could lead to the suspension of many fermentation processes, and even the death of microorganisms (Fei et al., 2011, Rodrigues and Pais, 2000, Royce et al., 2013). Considering the complexity and inhibition of the components of the EUOH, it is of great significance to study appropriate microbial treatment methods to realize the comprehensive and efficient utilization of organic components in EUOH.

In recent years, more and more attention has been paid to the treatment and high-value utilization of organic wastes by the lipid-producing fermentation process with oleaginous yeast (Cho and Park, 2018, Qin et al., 2017). Compared with the anaerobic fermentation process of producing biogas, ethanol, butanol, etc., lipid-producing fermentation is an aerobic fermentation process, which is much safer and easier to control. Besides, among various industrial microorganisms, oleaginous yeasts are relatively robust species and are easy to cultivate (Gao et al., 2017, Suutari et al., 1990). More importantly, oleaginous yeasts could use various types of low-cost substrates for cell growth and lipid synthesis, e.g. crude glycerol (Kamal et al., 2022), wastewater ( Chen et al., 2012; Wen and Li, 2021), lignocellulose biomass hydrolysate (Broos et al., 2022, Cianchetta et al., 2022, Wells et al., 2015), hydrophobic wastes (Patel and Matsakas, 2019), etc. It has been reported that a few oleaginous yeasts could take some kinds of five-carbon sugars and organic acids as carbon source (Li et al., 2020, Yu et al., 2014, Pereira et al., 2022). In addition, besides microbial lipids, yeast cells can also be used to produce polysaccharide products, which could be used as feed additives in poultry and livestock breeding (Kogan and Kocher, 2007, Li et al., 2022, Wu et al., 2021) or even play a role in anti-infective and antitumor therapy (Kogan et al., 2008, Qamar et al., 2022, Urazgaliev et al., 1992). However, for a variety of oleaginous yeasts that have been found, the fermentation performance varies with strains, and the fermentation characteristics of the same strain based on different substrates are also different (Gao et al., 2022, Zhang et al., 2022). Thus, for a specific fermentation substrate, the key to achieve good fermentation performances is to adopt appropriate strains to construct fermentation system.

The applications of microbial mixed culture, e.g. wastewater treatment, composting, and a broad spectrum of fermentative food preparations, are the historical foundation of current biotechnology. Nowadays, a targeted assembly of microorganisms to perform concerted bio-productions is forming a new cutting edge in biotechnology. Over the past decade, the research field of defined mixed cultures has gained increased attention due to their potential for process intensifification and the chance to produce unknown secondary metabolites (Finneran and Popovic, 2018, Schlembach et al., 2021). Especially, for low-cost substrates with complex components or containing inhibitors, synthetic mixed cultures or co-cultures has been reported to be one of the effective means to optimize the overall fermentation performance, because it can provide a more active, robust and stable microorganism community with comprehensive and beneficial metabolic characteristics (Cheirsilp et al., 2011, Wang et al., 2022). In recent years, the number of published studies on mixed-culture consolidated bioprocesses with low-cost substrates is increasing significantly. The products of these processes are, in many cases, acids, alcohols, lipids or enzymes for biomass pretreatment and hydrolysis (Schlembach et al., 2021, Schlembach et al., 2020, Zhao et al., 2018). In the field of microbial lipid production, more concerns have been oriented towards the co-culture systems of yeast and microalgae using low cost carbon substrates (Qin et al., 2017). Quite a few researchers have proposed various higher yield co-culture systems and analyzed the synergistic effects of oleaginous yeast and microaglae (Dias et al., 2019, Wang et al., 2022). However, the research on the mixed cultures of different oleaginous yeast is still far from enough. The existing studies on oleaginous yeasts for lipid production usually focuses on the screening of strains and the influence of substrate and fermentation conditions. Some of them adopted mixed-culture mode, but it is a pity that their discussions only stay in a simple evaluation of the overall yield, while there is still a lack of systematic and in-depth research on the specific function and influence of mixed strains fermentation compared with the single strain fermentation (Gao et al., 2022, Qin et al., 2017).

This present study mainly focused on the investigation and optimization of the fermentation performance of different oleaginous yeasts on Eucommia ulmoides Oliver hydrolysate (EUOH), more importantly, aims to explore and evaluate the function and influence of the mixed strains fermentation, compared with the single strain fermentation. First, the single strain fermentation performance of seven species of oleaginous yeasts on EUOH was investigated. Based on the metabolic capacity of these strains to the various sugars in EUOH and their cell growth and lipid production, four strains with different advantages were selected, including T. cutaneum and T. dermatis, which could metabolize all types of sugars in EUOH well and accumulate a large amount of biomass, and L. starkeyi and R. toruloides, which could reach a high lipid content in cells. Then, the selected strains with different advantages, in pairs with different combinations, were used for the mixed strains fermentation on EUOH. Through systematic investigations of substrate metabolism, growth and lipid production, COD and ammonia-nitrogen removals in all the mixed strains fermentations, as well as further studies on the change of yeast polysaccharide, the function and effect of the mixed strains fermentation (compared with the single strain fermentation) were analyzed and evaluated.

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