Ethanol and acetate production from synthesis gas using Clostridium Langali bacteria

Dissertation of the Ph.D. course in the field of chemical engineering, biotechnology orientation

Abstract

Clostridium langalli is a highly anaerobic aestogenic bacterium that can grow on the components of synthesis gas, i.e. CO and H2/CO2, and convert them into ethanol and acetate at ambient temperature and pressure. During this process, the bacterium shows a complex metabolic pathway that includes both estogenic (acid production) and salontogenic (solvent production) phases. In the process of heterotrophic growth of this bacterium, the effect of different organic substrates (fructose, glucose, ethanol and acetate) on the beginning of the metabolic shift towards the alcohol production phase was investigated. The results of the discontinuous fermentation process showed that the use of fructose as an organic substrate led to the production of the same molar ratio of ethanol (27.1 mmol/L) and acetate (26.3 mmol/L). In the process of autotrophic growth of bacteria with synthesis gas, in order to reduce the reducing potential of the culture medium and change the direction of the flow of electrons towards the alcohol production phase, different reducing solutions (sodium sulfide and/or acidic cysteine ??with different concentrations) were used at different initial pH (6.8 or 5.9) of the culture medium in discontinuous bioreactors. The highest molar ratio of ethanol to acetate production (0.65) was obtained in the culture medium containing 5.07 mmol/l acidic cysteine ??and at the initial pH of 5.9, which was probably related to the presence of more electrons in this medium. To determine the biokinetic parameters related to the growth rate, substrate consumption and product production, synthesis gas fermentation process was carried out in discontinuous bioreactors with different synthesis gas pressures. To describe the kinetics of bacterial growth rate on synthesis gas components (CO and H2), a kinetic growth model was developed based on a binary substrate using Long's model for CO and Monod's model for H2. This model could also predict the inhibitory effects of CO at high pressures on cell growth. Modified Veltra, Andrew, and Gomperts kinetic models were also used to describe cell growth, substrate consumption, and product production. The continuous process of synthesis gas fermentation was carried out in a two-liter stirred bioreactor. The effect of various operational parameters such as liquid dilution rate, synthesis gas flow intensity into the bioreactor and stirrer speed on the performance of the culture medium was investigated. The highest specific production rate (0.0048 mol per gram of cells per hour), product yield (0.178 mol of product per mole of substrate) and the molar ratio of ethanol to acetate production of 0.73 (with 30 and 41 mmol per liter of ethanol and acetate) were obtained at the liquid dilution rate of 0.018 (per hour), gas flow intensity of 12 (ml/min) and stirring speed of 500 (rpm). 

Key words

 Ethanol, acetate, Clostridium Langali, synthesis gas fermentation

1         Chapter 1: Introduction

rtl;">1-1      Introduction

Since the beginning of the 20th century, the production of fuel and chemical compounds from syngas as a method for producing renewable fuels has attracted the attention of scientific and industrial communities. However, most of the advances and discoveries that have been made in this field are related to the use of catalytic processes and metal-based catalysts. Recently, the attention of researchers has been directed to the production of biological fuels and chemical compounds from synthesis gas through biological methods, because the use of microbes as biocatalysts has advantages over metal-based catalysts.

Today, many efforts are made to produce biological fuels, but this issue has become one of the controversial issues in the scientific and human societies. The production of first generation biofuels from food sources is considered unethical and has always been criticized due to the urgent need of many countries for food. Synthesis gas fermentation for the production of second-generation biofuels [2] can answer most of the criticisms regarding the production of fuel from food products. The production of second-generation biofuels from non-food sources, generally agricultural waste and organic waste, includes two basic technologies, in which biomass is first converted into gas, and then the synthesis gas produced as a substrate in a microbial or catalytic process is converted into biological fuel.

Despite the studies and researches that have recently been conducted on the synthesis gas fermentation process as a sustainable and renewable method for the production of biological fuels, this process is still considered an undeveloped technology and it is necessary to overcome various technical and economic challenges before the commercialization of this process. 

1-2     Biofuels

The global production of biofuels has increased rapidly in the last decade, but this growing industry has brought with it important concerns. First generation biofuels are produced from primary food sources such as starch, sugar, vegetable oils and animal fats. Although the production of first-generation biofuels, such as the production of ethanol from corn in the United States, ethanol from sugarcane in Brazil, and biodiesel from rapeseed and sunflower in Europe, continues as commercialized processes, despite many criticisms regarding the sustainability of these fuels and their competition with food production, second-generation biofuels have received much attention [1]. Second generation biofuels are produced from lignocellulosic biomass [3] that are not a food source. An overview of the primary sources used to produce second-generation biofuels is presented in Figure 1-1 [1, 2]. In general, these raw materials are divided into agricultural waste, organic waste, and biomasses that grow rapidly and are cultivated for energy production [4]. Therefore, the second generation biofuels have advantages such as the use of waste and residues and the use of barren lands, especially in rural areas. However, if the production of these fuels competes with food products on the available land, the suitability of these fuels will be doubted in terms of production sustainability. There is another concern in this field that the indiscriminate harvesting of agricultural waste to produce fuel and biological energy will have a negative impact on soil fertility and its quality [3]. In this process, the bacterium presents a complex metabolic pathway including both the acetogenic and solventogenic phases. During the heterotrophic batch cultivation of the bacterium, the effect of various carbon sources (fructose, glucose, ethanol and acetate) on triggering the metabolic shift towards solventogenesis was considered. The fermentation results demonstrated the equimolar production of ethanol (27.1 mM) and acetate (26.3 mM) in the presence of fructose. During the autotrophic growth of the bacterium with synthesis gas for lowering the redox potential of the growth medium and alteration of the electron flow towards solventogenesis, various reducing solutions (sodium sulfide and/or cysteine-HCl with various concentrations) at different initial medium pH (6.8 or 5.9) were used in the batch bioreactors. The results suggested the plausible provision of more electrons into the culture in the presence of 5.07 mM cysteine-HCl at the medium pH 5.9, as a promoted ethanol to acetate molar ratio of 0.65 was achieved in this culture. In order to determine the intrinsic growth, substrate uptake and product formation biokinetic parameters in synthesis gas fermentation process, the bacterium was grown in various pressurized batch bioreactors. A dual-substrate growth kinetic model using Loung for CO and Monod for H2 was developed to describe the growth rate kinetics of the bacteria on these substrates. This model also accommodated CO inhibitory effects on cell growth at high CO partial pressures. The Volterra model, Andrews and modified Gompertz were respectively adopted to describe the cell growth, substrate uptake rate and product formation. Continuous synthesis gas fermentation experiments were carried out in a 2.0 L CSTR bioreactor.