Identification of Inhibitors in Lignocellulosic Slurries and Determination of Their Effect on Hydrocarbon-Producing Microorganisms.

Shihui Yang,Qing Yang,Min Zhang,Philip T Pienkos,Yat-Chen Chou,Mary Ann Franden

doi:10.3389/fbioe.2018.00023

Abstract

The aim of this work was to identify inhibitors in pretreated lignocellulosic slurries, evaluate high-throughput screening strategies, and investigate the impact of inhibitors on potential hydrocarbon-producing microorganisms. Compounds present in slurries that could inhibit microbial growth were identified through a detailed analysis of saccharified slurries by applying a combination of approaches of high-performance liquid chromatography, GC-MS, LC-DAD-MS, and ICP-MS. Several high-throughput assays were then evaluated to generate toxicity profiles. Our results demonstrated that Bioscreen C was useful for analyzing bacterial toxicity but not for yeast. AlamarBlue reduction assay can be a useful high-throughput assay for both bacterial and yeast strains as long as medium components do not interfere with fluorescence measurements. In addition, this work identified two major inhibitors (furfural and ammonium acetate) for three potential hydrocarbon-producing bacterial species that include Escherichia coli, Cupriavidus necator, and Rhodococcus opacus PD630, which are also the primary inhibitors for ethanologens. This study was strived to establish a pipeline to quantify inhibitory compounds in biomass slurries and high-throughput approaches to investigate the effect of inhibitors on microbial biocatalysts, which can be applied for various biomass slurries or hydrolyzates generated through different pretreatment and enzymatic hydrolysis processes or different microbial candidates.

Highlights

Renewable energy has attracted increasing interest because of the concern for the high demand on fossil fuels and the contribution of burning fossil fuels to global climate change
The standard analysis of carbohydrates and acids within saccharified slurries using high-performance liquid chromatography (HPLC) method indicated that acetate and furfural were higher in the CS material than in the DCS material (Table 1)
Furfural and acetate are the most toxic compounds among chemicals tested and all species are sensitive to them, with furfural being more toxic than acetate (Table 3), indicating that pretreatment severity must be minimized even if aerobic organisms are to be used for hydrocarbon biofuel production

Summary

Introduction

Renewable energy has attracted increasing interest because of the concern for the high demand on fossil fuels and the contribution of burning fossil fuels to global climate change. Lignocellulosic biomass represents an abundant renewable bioresource for the production of biofuels and biochemicals (Kurosawa et al, 2013; Laskar et al, 2013; Xie et al, 2013), and its enhanced use would address several societal and economic issues. Pretreatment and enzymatic hydrolysis (EH) are, required to release mono-sugars from biomass for biochemical conversion. Efforts have been carried out at bench and pilot scales to discover novel cellulolytic enzymes, improve enzyme cocktail formula such as the inclusion of accessory enzymes or surfactants (Romaní et al, 2014a,b; Agrawal et al, 2015a,b, 2017), the impact of inhibitors generated during above pretreatment on cellulolytic enzymes and the requirement of high enzyme loading during subsequent EH affects the economic bioconversion of lignocellulosic biomass

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