Consolidated bioprocessing of cassava starch into microbial lipid for biodiesel production by the amylolytic yeast Lipomyces starkeyi

https://doi.org/10.1016/j.indcrop.2022.114534Get rights and content

Highlights

  • CBP of cassava starch into lipid was promising by the amylolytic yeast L. starkeyi.

  • Lipid titer and lipid yield reached 13.99 g/L and 0.187 g/g, respectively.

  • CBP was superior to SHF with simpler process, lower cost, and higher oleaginicity.

  • Starch hydrolysis and lipogenesis were assessed using a small-scale metabolic model.

  • Biodiesel prepared from the CBP-derived lipids showed high-quality fuel properties.

Abstract

Direct conversion of starch materials into lipid has been rarely reported as most oleaginous species are unable to efficiently produce amylolytic enzymes. Herein, consolidated bioprocessing (CBP) of cassava starch was described efficiently for lipogenesis by Lipomyces starkeyi. Lipid concentration and yield reached 13.99 g/L and 0.187 g/g, respectively, from 75 g/L cassava starch under optimal conditions. The CBP strategy featured zero enzyme cost, simpler process, and higher oleaginicity compared with the separate hydrolysis and fermentation (SHF) strategy. Amylolytic enzymes with α-amylase, maltase and glucoamylase activities were secreted into extracellular space and the amylase activity reached 3.65 U/mL as maximum. The genome of L. starkeyi was sequenced and a small-scale metabolic model was established. The mechanism of starch hydrolysis for lipogenesis by L. starkeyi was estimated accordingly. The estimated biodiesel parameters of the CBP-derived lipid samples indicated high-quality fuel properties. The CBP strategy provides a highly integrated and techno-economic route for starch-to-lipid conversion.

Introduction

Microbial lipids have been regarded as potential alternative contenders for sustainable biodiesel production and substitutes for high-value nutritional products (Papanikolaou and Aggelis, 2011, Ratledge and Wynn, 2002). Lipid generated from a series of low-cost materials including lignocellulosic biomass by oleaginous species has garnered widespread attention in the past decades (Jin et al., 2015, Qadeer et al., 2017). Lignocellulosic sugars have been verified suitable for lipid synthesis (Gong et al., 2012, Liu et al., 2021). Nevertheless, the structure recalcitrance of lignocelluloses severely impedes the release of monomeric sugars (Himmel et al., 2007). The lignocellulose-to-lipid roadmap routinely involves in three steps, namely pretreatment to disrupt the rigid structure of lignocellulosic biomass, enzymatic hydrolysis to release sugar mixture, and fermentation to convert the lignocellulosic sugars into lipid by oleaginous microorganisms. Costly pretreatment and enzymatic hydrolysis processes are the two primary obstacles hindering the commercial success.

To reduce the costs, it is crucial to explore low-cost feedstocks particularly liable to saccharification and to develop integrated processes. Cassava is an important starch-rich plant widely planted in barren land and harsh climates worldwide (Lin et al., 2019). It is a comparatively cheap non-grain crop in some countries including China. In addition, tremendous amounts of starchy wastes including cassava pulp and starch wastewater are generated (Xue et al., 2010, Padi and Chimphango, 2020). For example, cassava pulp, a side product of starch industry in tropical areas without scientific processing, routinely contains large amount of residual starch (Tanimura et al., 2014). Starch-to-lipid process commonly involves in two steps, namely enzymatic degradation of starch into fermentable sugars and bioconversion of the resultant sugars into microbial lipid. Compared with the lignocellulose-to-lipid routes, this process is more advantageous due to no need of pretreatment, no generation of inhibitory compounds, and relatively lower enzyme cost.

The hydrolysates of cassava starch have been effectively applied for lipid fermentation by some oleaginous yeasts including Rhodosporidium toruloides and Rhodotorula mucilaginosa (Li et al., 2010, Wang et al., 2012). However, starch could not be directly metabolized for lipid overproduction as these oleaginous species are unable to efficiently produce extracellular amylolytic enzymes. A saccharification process is obligatorily required for starch hydrolysis before lipid fermentation (Schneider et al., 2013). It should be noted that the saccharification process is generally complicated and costly. The starch is routinely liquefied by α-amylase and then saccharified by glucoamylase, which necessitates high input of energy (Cripwell et al., 2020).

To solve these problems, the immobilized amylolytic yeast Saccharomycopsis fibuligera and the oleaginous yeast R. toruloides were co-cultured to convert cassava starch into microbial lipid in one-step (Gen et al., 2014). Consolidated bioprocessing (CBP) of starchy materials into ethanol and butanol has been realized using microorganisms featuring amylolytic enzymes secretion capacity (Cripwell et al., 2020, Li et al., 2016, Lin et al., 2019, Roble et al., 2003). Interestingly, some oleaginous species with amylases secretion ability have been evaluated the potential to integrate the hydrolysis and fermentation processes and minimize the enzyme cost (Chaturvedi et al., 2018, Chaturvedi et al., 2019, Muniraj et al., 2015, Tanimura et al., 2014, Wild et al., 2010). It is worth mentioning that Tanimura and co-workers have assessed starch-assimilation capacity of 1200 yeasts. Among them, Cryptococcus terricola was observed the optimal strain with a maximum lipid content of 61.96% from 5% soluble starch (Tanimura et al., 2014). However, lipid concentration was merely 3.02 g/L with almost half of the starch not hydrolyzed although the CBP was held for 10 days. Lipomyces starkeyi has been served as a potential candidate for α-amylase production (Kang et al., 2004, Kelly et al., 1985). Lipid content of 40% and lipid yield of 0.16 g/g were obtained when L. starkeyi NRRL Y-11557 was cultured on 27.3 g/L of soluble sweet potato starch (Wild et al., 2010).

CBP is a promising strategy due to the outstanding potential for cost reduction (Lynd et al., 2005). L. starkeyi is an excellent lipid producer featuring wide substrate spectrum, excellent fatty acid composition for high-quality biodiesel, and negligible lipid remobilization (McNeil and Stuart, 2018, Sutanto et al., 2018). In the present study, a CBP strategy that integrates the amylolytic enzymes production, enzymatic hydrolysis of cassava starch, and lipid fermentation in one-step was described by the amylolytic strain L. starkeyi AS 2.1560. The effects of pH, carbon/nitrogen (C/N) molar ratio, and initial cassava starch concentration on lipid production were preliminarily investigated. The extracellular amylase activity was analyzed and the hydrolysis capacity was evaluated. The genome of L. starkeyi was sequenced and a simplified metabolic model was established to assess the capacities of starch hydrolysis and lipid biosynthesis. The CBP of cassava starch provides a simplified and techno-economic way for starch-to-lipid conversion.

Section snippets

Strain and media

A variety of oleaginous yeasts applied in this study included L. starkeyi (AS 2.1560, NRRL Y-11557), Trichosporon cutaneum (CGMCC 2.3013, GIM 2.67), Trichosporon mucoides (CICC 1368), Cutaneotrichosporon oleaginosum (ATCC 20509), and Yarrowia lipolytica (po1g). These yeasts were maintained on yeast peptone dextrose (YPD) plates with subculturing twice a month. The YPD medium was composed of 20 g/L glucose, 10 g/L yeast extract, 10 g/L peptone, and 18 g/L agar.

General procedure for the CBP of cassava starch

Unless mentioned elsewhere, the

Lipid production on cassava starch by different oleaginous yeasts through CBP strategy

Seven oleaginous yeasts were preliminarily assessed for lipogenesis from 50 g/L cassava starch through CBP strategy. The culture was terminated at 72 h and the lipid production was compared. It was clear that extremely low lipid concentrations varying from 0.8 to 1.3 g/L were observed for T. cutaneum, T. mucoides, C. oleaginosum, and Y. lipolytica ( Fig. 1). It was suggested that these yeasts showed poor abilities to hydrolyze starch into fermentable sugars for lipogenesis. Interestingly, L.

Conclusions

CBP of cassava starch was found very promising for the oleaginicity of the amylolytic yeast L. starkeyi. The amylolytic enzymes were induced and secreted into the extracellular space to efficiently hydrolyze of cassava starch into fermentable sugars. High lipid concentration and yield were realized with the complete assimilation of the released sugars. The CBP strategy featured zero enzyme cost, simpler process, and higher oleaginicity compared with the SHF strategy. The biodiesel prepared

CRediT authorship contribution statement

Junlu Zhang: Investigation, Software, Writing – original draft. Yanan Wang: Resources, Investigation, Software. Qingling Gou: Investigation, Formal analysis. Wei Zhou: Software, Validation. Yantao Liu: Data curation. Jikun Xu: Investigation. Yi Liu: Formal analysis. Wenting Zhou: Conceptualization, Methodology, Writing – original draft. Zhiwei Gong: Supervision, Project administration, Conceptualization, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was financially supported by the National Natural Science Foundation of China (51608400), the Fundamental Research Funds for the Central Public Welfare Research Institutes (ZZ13-YQ-085-C1), and the Fund from Key Laboratory of Hubei Province for Coal Conversion and New Carbon Materials (WKDM202108).

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    These authors contributed equally to this work.

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