Improved n-butanol production from lignocellulosic hydrolysate by Clostridium strain screening and culture-medium optimization

Highlights

• Sugarcane straw hydrolysate was studied as feedstock for ABE fermentation.

• ABE fermentation with hydrolysed medium were compared in twelve Clostridium strains.

• Optimization of cultivation parameters improved butanol and hydrolysate tolerance.

• 11 g/L of solvents were produced from 79% of pure lignocellulosic hydrolysate medium.

Abstract

The use of lignocellulosic hydrolysate is a promising strategy to make fuel and chemical production feasible through biological processes. The major challenges facing this process are the presence of inhibitors in hydrolysate, as furan derivatives and phenolic compounds, and the inefficient consumption of the xylose found in hydrolysate. Therefore, this study aimed to select ideal Clostridium strains and an optimal medium for microbiological production of n-butanol from sugarcane straw hydrolysate with high yield and productivity. From a screening of twelve Clostridium strains, two strains stood out: Clostridium saccharoperbutylacetonicum DSM 14923, by its potential to produce high titer of n-butanol (4.95 g/L from 11.6 g/L glucose and 3.6 g/L xylose), and Clostridium saccharobutylicum DSM 13864, by its capacity to concomitantly consume similar quantities of glucose and xylose. Optimization of culture medium and inoculum preparation allowed an improvement in n-butanol production from 0.65 g/L for 5.39 g/L. Moreover, with optimized medium, a higher tolerance to lignocellulosic hydrolysate was obtained, avoiding a premature end to fermentation at hydrolysate and enabling the cultivation of these strains in 79% pure lignocellulosic hydrolysate. Among all the strains tested for lignocellulosic hydrolysate fermentation, C. saccharobutylicum DSM 13864 has the best performance. This strain, in a culture medium composed by 79% of pure lignocellulosic hydrolysate, showed a consumption of 95% of all sugar available (34 g/L of glucose and 22 g/L of xylose) and a total acetone, n-butanol, and ethanol (ABE) production of 10.33 g/L, being the most suitable strain for n-butanol production using this substrate.

Introduction

In recent years, growing concerns about the environment and the instability of oil prices has prompted studies regarding the production of fuels and chemicals through biological processes. Among the many compounds that can be produced by microorganisms, butanol stands out for its importance in chemical industries. Among butanol isomers, isobutanol has the potential to be used as fuel, showing better features than ethanol for this purpose [1], [2]. While n-butanol isomer is an important bulk chemical for industrial purposes that is applied mainly for the generation of acrylate, methacrylate esters, butyl acetate, and butyl glycol. It is also used for the production of a variety of cosmetics, antibiotics, hormones, vitamins, hydraulic and brake fluids, lubricants, flotation aids, and products for the leather and paper industries [3]. Unlike isobutanol, which is mainly petrochemically produced, n-butanol can be produced through fermentation by Clostridium species [4].

The genus Clostridium includes gram-positive, anaerobic, spore-forming bacteria, and it comprises pathogenic and solventogenic strains. The solventogenic strains are responsible for a process known as acetone-butanol-ethanol (ABE) fermentation that results in the production of acetone, n-butanol, and ethanol. The Clostridium metabolic process for n-butanol production can be divided in two sequential phases: acidogenesis and solventogenesis. In the first stage, which happens in the exponential growth phase, the principal products are butyric acid, acetic acid, hydrogen, and carbon dioxide. The second stage, solventogenesis, takes place at the end of the exponential phase and during the stationary phase, and is characterized by the re-assimilation of acids with a concomitant conversion into the solvents acetone, n-butanol, and ethanol [5].

ABE fermentation is an old industrial fermentation process and was the main source of n-butanol for many years during the early 20th century. However, due to economic factors such as the high cost of fermentation substrate and the progress of the petrochemical industries, n-butanol production by fermentation was replaced with petrochemical routes [3]. Looking for the re-establishment of ABE fermentation, many approaches involving different metabolic engineering strategies are being conducted for an improvement at n-butanol production by Clostridium strains. As promising strategies we can cited the use of ClosTron system [6], [7] and the CRISPR system [8], [9], genetic tools that allow the creation of mutants and, consequently, open new avenues for research on metabolic engineering. However, despite advances in metabolic engineering, other strategies need to be addressed in order to achieve an economically viable ABE process. In recent years, mostly due to environmental and economic issues related to oil use, one of these strategies that has been studied is the use of lignocellulosic biomass as feedstock.

Besides contributing to reduced greenhouse gas emissions, lignocellulosic biomass is an abundant and cheap substrate for fermentation, being a good alternative to the typical highly priced fermentation substrate. Moreover, its sugar does not compete with food crops and has higher yield of dry mass per hectare than the conventional sugarcane [10], [11]. The main constituents of lignocellulosic matrix are: cellulose, formed by ligated glucoses; hemicellulose, formed by ligated galactose, glucose, mannose, xylose, and arabinose; and lignin, a complex phenolic polymer [12]. Before it can be used as a fermentation substrate, lignocellulosic material must pass through two stages: pretreatment and hydrolysis. Pretreatment breaks down the tight structure of lignocellulose, increasing the material's surface area and facilitating the hydrolysis of polymeric sugars, cellulose and hemicellulose, into monomers. There are different technologies of biomass pretreatment, including chemicals (acid, alkaline, oxidative delignification, and organosolv), physicals (milling, pyrolysis, and microwave), physiochemicals (steam-explosion, ammonia fiber expansion, and CO2 explosion) and biological (degradation of lignocellulosic material by fungi and bacteria) [10]. After pretreatment, a large fraction of sugar remains in the form of polysaccharides and oligosaccharides, which will be broken down into sugar monomers during hydrolysis. These hydrolysis of polymeric sugars can be performed using acids, enzymes, or a combination of both [12]. According to the technology used for biomass treatment some undesirable degradation compounds can be produced, such as furan derivatives (furfural, 5-hydroxymethyl-furfural (HMF)), aliphatic acids (acetic, formic, and levulinic acids) and phenolic compounds (chlorogenic acid, 4-hydroxybenzaldehyde, vanillic acid, caffeic acid, syringic acid, vanillin, syringaldehyde, p-coumaric acid, acetosyringone, ferulic acid, and 4-hydroxybutyl acrylate). All these degradation compounds are known to negatively affect some microbial fermentation processes, including the ABE fermentation [12], [3]. Considering these points, this study aimed to establish an optimised fermentation conditions to produce n-butanol using an industrial lignocellulosic hydrolysate and a high n-butanol-producer strain. Therefore, this work performed a screening of Clostridium strains suitable for n-butanol production using sugarcane straw lignocellulosic hydrolysate as a substrate. The fermentation profiles of the selected strains were determined, followed by an optimisation of cultivation conditions in lignocellulosic hydrolysate to increase n-butanol production and hydrolysate tolerance.