Continuous cellulosic bioethanol co-fermentation by immobilized Zymomonas mobilis and suspended Pichia stipitis in a two-stage process

Highlights

• Continuous production of bioethanol from pretreated sugarcane bagasse was studied.

• Continuous separate hydrolysis and co-fermentation process was studied.

• Simultaneous saccharification and co-fermentation process was compared.

• Highest yield 0.414 g/g and highest productivity 1.868 g/L/h were separately reported.

Abstract

Bioethanol produced from lignocellulosic materials has been considered as one of the most promising fuels to replace fossil fuels. Immobilized yeasts or bacteria have been frequently used in continuous system due to its feasibility for repeated use with high biomass retention during the continuous process. In this study, continuous SHcF (separate hydrolysis and co-fermentation) and SScF (simultaneous saccharification and co-fermentation) were evaluated for ethanol production from alkaline pretreated sugarcane bagasse using Zymomonas mobilis (PVA immobilized cells) and Pichia stipitis (suspended cells). In SHcF fermentation, the ethanol yield and productivity of 0.36 g ethanol/g cellulose (corresponding to 70.65% of theoretical yield) and 1.868 g/L/h were achieved. In contrast, SScF system resulted in an ethanol yield of 0.414 g ethanol/g cellulose (corresponding to 81.17% of theoretical yield) and ethanol productivity of 0.705 g/L/h. The performance of the two systems are compared and discussed.

Introduction

Bioethanol is one of the most potential biofuels that can replace fossil fuels. Bioethanol has some advantages as fuel as it produces less CO, has higher octane number, and has higher heat of vaporization than the gasoline [1], [2], [3]. Alternatively, using ethanol-blended fuel for automobiles can significantly reduce petroleum use and exhaust greenhouse gas emission [4], [5]. Ethanol is currently used as gasoline additive in the European Union and the United States (E10 and allowed up to E85). This has reduced emission of GHGs by 10% and is fast approaching the proposed 20% reduction scenario proposed by the European Biofuels Technology Platform (2011). Until now, most of the bioethanol is still produced from food-based crops such as sugarcane and maize. The utilization of these raw materials is rather simple, but it can conflict with the food supply and arable lands [6]. On the other hand, lignocellulose is one of the planet’s most abundant form of biomass [7], and also available in low cost [8]. Therefore, lignocellulosic-based bioethanol could be a potential substitute for gasoline in the near future [9], [10].

Raw lignocellulosic materials are mainly composed of cellulose, a glucose polymer; hemicellulose, a heterogeneous polymer composed of glucose, mannose, xylose and arabinose; bound together by lignin, a highly branched and substituted mononuclear aromatic polymer [11], [12]. The specific proportion of the components vary with the source material and pretreatment is a necessary step to open up the cellulose and hemicellulose for hydrolysis [13]. Suagrcane bagasse is the main by-product obtained after extraction of sugarcane juice and is produced in large quantities by the alcohol and sugar industries in Brazil, Indonesia, India and China [14]. Abundance of this bagasse lead to its use as a renewable feedstock in bioethanol production. The pretreatmets generally used for sugarcane bagasse hydrolysis are acid and alkaline methods. Acid pretreatments solubilize hemicellulose and disrupts lignin structure whereas alkaline pretreatment solubilizes lignin and enhance enzymatic hydrolysis [11]. It is also reported that alkaline pretreatment causes less sugar degradation than acid pretreatment and it was shown to be more effective on wood materials [15]. In addition, alkaline pretreatment can significantly remove acetyl group from hemicellulose, which lowers steric hindrance for enzyme activity [16]. Hydrolysis process can be applied after pretreatment process to hydrolyze the pretreated lignocellulose to yield monosaccharide sugars. There are at least three methods of hydrolysis, including dilute acid hydrolysis, concentrated acid hydrolysis, and enzymatic hydrolysis. At present, the prevalent technique for cellulosic ethanol production is an enzyme-based process because it is more environmental-friendly and gives higher hydrolysis yield than acid hydrolysis [17]. Regardless, pretreatment and hydrolysis steps have been highlighted as the most cost incurring steps in the bioprocessing of lignocellulosic materials [18].

The hydrolysis of sugarcane bagasse, like many other lignocellulosic biomass, release a mix of hexoses and pentoses that include glucose, mannose, galactose, xylose and arabinose. Glucose and xylose are the most dominant sugars and the major bottleneck in cost effective bioethanol production is the efficient fermentation of both these sugars. The traditional ethanol fermenters, S. cerevisiae and Z. mobilis cannot utilize pentoses, but can only ferment glucose to ethanol. Pichia stipitis, Candida shehatae and Pachysolen tannophilus are the major pentose fermenting yeasts that have been used extensively. Over the years, researchers have adapted various technologies that include co-culture of hexose and pentose metabolizing strains, genetic engineering of microorganisms to ferment both sugars and so on. The co-culture systems for efficient fermentation of the mixed sugars has been reviewed elsewhere [19].

Zymomonas mobilis is an obligatory fermentative microorganism which utilizes sucrose, glucose, and fructose via Entner-Doudoroff (ED) pathway, which is used mostly by strictly aerobic organisms such as Pseudomonas [20]. This pathway produces only one ATP, compared to two ATP produced by EMP pathway, enabling Z. mobilis to produce less biomass and more ethanol when compared to S. cerevisiae [21], [22]. In addition, its specific ethanol productivity is up to five times higher than S. cerevisiae and its ethanol yield is also 5% higher than S. cerevisiae, and it can tolerate ethanol up to 120 g/L [23], [22], making this bacterium a potential candidate for ethanol fermentation from glucose. Pichia stipitis (recently reclassified as Scheffersomyces stipitis) is one of the few microorganisms that can naturally produce ethanol from xylose [24]. It can ferment xylose quite effectively, nearly reaching up to 67% of theoretical ethanol yield [25]. It can also ferment glucose, galactose, cellobiose, and xylan [24], [22], with the ability to ferment the latter two due to its natural ability to produce β-glucosidase and xylanase [26]. Its ability to utilize xylose to produce ethanol makes this yeast a potential candidate for ethanol fermentation from xylose. Z. mobilis and P. stipitis has already been used in a co-culture process but it was found that viable cells of Z. mobilis inhibited xylose fermentation by P. stipitis [27]. The authors also suggested the spatial separation of the cells as a prerequisite for successful fermentation of the mixed sugars. This was achieved by the design of a special fermenter and immobilized Z. mobilis [27]. Singh et al developed a sequential co-culture system for P. stipitis and Z. mobilis and showed ethanol production from both biomass hydrolysate and synthetic sugars [28]. A continuous culture with two separate bioreactors for Z. mobilis and P. stipitis was also developed and ethanol production was demonstrated from biomass hydrolysate and synthetic medium [29].

Continuous system has an advantage in terms of higher productivity over the batch system. Despite this advantage, most of the ethanol fermentation studies are investigated using batch process [30]. The difficulty of controlling the dilution rate to match cell growth is the primary reason of choosing batch system over continuous system. However, this problem can be overcome by deploying immobilized cell system. Immobilization of live cells can be advantageous, particularly in continuous systems because of their increased stability, reusability and increase in volumetric productivity. Also, membrane bioreactor can be used so that the cell can be kept inside the system to prevent cell washout in continuous process [31].

In this study, alkaline pretreated bagasse was used for continuous cellulosic ethanol production in two stage SHF (separate hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation) by PVA-immobilized Zymomonas mobilis and suspended Pichia stipitis. The two stage process ensures effective glucose utilization with high yield ethanol production by Z. mobilis and ethanol production from pentoses in the P. stipitis system. This ensures complete utilization of all the lignocellulosic sugars and high yield ethanol fermentation. SSF and SHF were evaluated for ethanol fermentation performance, lignocellulose conversion efficiency to arrive at an efficient two-stage ethanol fermenting system which carries out cellulose hydrolysis, hexose fermentation and pentose fermentation.