Injection of air into the headspace improves fermentation of phosphoric acid pretreated sugarcane bagasse by Escherichia coli MM170

Abstract

Microaeration (injecting air into the headspace) improved the fermentation of hemicellulose hydrolysates obtained from the phosphoric acid pretreatment of sugarcane bagasse at 170 °C for 10 min. In addition, with 10% slurries of phosphoric acid pretreated bagasse (180 °C, 10 min), air injection into the headspace promoted xylose utilization and increased ethanol yields from 0.16 to 0.20 g ethanol/g bagasse dry weight using a liquefaction plus simultaneous saccharification and co-fermentation process (L+SScF). This process was scaled up to 80 L using slurries of acid pretreated bagasse (96 h incubation; 0.6 L of air/min into the headspace) with ethanol yields of 312–347 L (82–92 gal) per tonne (dry matter), corresponding to 0.25 and 0.27 g/g bagasse (dry weight). Injection of small amounts of air into the headspace may provide a convenient alternative to subsurface sparging that avoids problems of foaming, sparger hygiene, flotation of particulates, and phase separation.

Introduction

Government initiatives in Europe (Strategic Energy Technology Plan), the United States (Advance Energy Initiative), and elsewhere are promoting the development of lignocellulosic feedstocks for renewable chemicals and fuels. Between 50% and 75% of lignocellulosic biomass is composed of two carbohydrate polymers, cellulose and hemicellulose. After conversion to sugar monomers, biocatalysts can be used to transform these carbohydrates into fuels, building block molecules for plastics, and other compounds currently made from petroleum.

Significant progress has been made in the pretreatment process during the past few years resulting in an increase in sugar yields and a reduction in the levels of inhibitors related to the degradation of sugars and lignin (Kumar et al., 2009). However, most pretreatment options require additional steps to detoxify the pretreated biomass slurry, contributing to the high processing costs and hindering successful commercialization (Lynd et al., 2008). Several methods have been used to neutralize the toxicity of the side products formed by dilute acid pretreatment including overliming, organic solvent extraction, ion exchange resins, and reducing agents (Barbosa et al., 1992, Frazer and McCaskey, 1989, Leonard and Hajny, 1945, Martinez et al., 2001). More recently, Alriksson et al. (2011) reported the use of sulfur compounds for the in situ detoxification of wood and sugarcane bagasse hydrolysates. Nieves et al. (2011) used sodium metabisulfite to increase the ethanol yield and reduce the time of fermentation for dilute acid (phosphoric) pretreated sugarcane bagasse using ethanologenic Escherichia coli. Geddes et al. (2011) successfully fermented dilute acid pretreated sugarcane bagasse using a hydrolysate-resistant E. coli biocatalyst without the need for detoxification. Using these resistant biocatalysts, a simplified fermentation process was developed based on corn dry milling that includes dilute phosphoric acid pretreatment, partial liquefaction, and fermentation of hemicellulose syrups simultaneously with cellulose in a single vessel, L+SScF (liquefaction plus simultaneous saccharification and co-fermentation of cellulose and hemicellulose sugars) (Geddes et al., 2011).

The addition of air during fermentation has previously been investigated as a strategy to improve ethanol fermentation with yeast and bacteria. Initially, sparging with air was used to increase the cell concentration of yeast inoculums to overcome the toxins in hydrolysates of wood (Leonard and Hajny, 1945). However, mixed results have been reported for air sparging during other yeast fermentations (Alfenore et al., 2004, Ghaly and El-Taweel, 1995, Schirmer-Michel et al., 2009, Zhang et al., 2010). A series of publications from G. T. Tsao’s lab at Purdue University identified the optimum level of oxygen for 2,3-butanediol production by Klebsiella oxytoca (Beronio and Tsao, 1993, Jansen et al., 1984). Lawford and Rousseau (1994) tested different aeration rates during ethanol fermentation with recombinant E. coli and reported that subsurface sparging decreased ethanol yield, increased cell yield, and increased alternative fermentation products. Okuda et al. (2007) later demonstrated that subsurface sparging with low levels of air (microaeration) could be used to increase ethanol production from dilute acid hydrolysates (hemicellulose) using ethanologenic E. coli. Sanny et al. (2010) recently demonstrated that expression of Vitreoscilla hemoglobin can be used to enhance the efficiency of low oxygen utilization and ethanol production by ethanogenic strains of E. coli.

In this paper we have investigated the effectiveness of supplying small amounts of air into the headspace during the fermentation of dilute acid hydrolysates of sugarcane bagasse and during the fermentation of acid pretreated bagasse slurries (10% dry weight) using ethanologenic E. coli MM170 and the L+SScF process. Fermentations with slurries of dilute acid pretreated sugarcane bagasse were successfully scaled up to 80 L.