Production of ethanol from thin stillage by metabolically engineered Escherichia coli
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- Gonzalez, R., Campbell, P. & Wong, M. Biotechnol Lett (2010) 32: 405. doi:10.1007/s10529-009-0159-2
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Thin stillage is a by-product generated in large amounts during the production of ethanol that is rich in carbon sources like glycerol, glucose and maltose. Unfortunately, the fermentation of thin stillage results in a mixture of organic acids and ethanol and minimum utilization of glycerol, the latter a compound that can represent up to 80% of the available substrates in this stream. We report here the efficient production of ethanol from thin stillage by a metabolically engineered strain of Escherichia coli. Simultaneous utilization of glycerol and sugars was achieved by overexpressing either the fermentative or the respiratory glycerol-utilization pathway. However, amplification of the fermentative pathway (encoded by gldA and dhaKLM) led to more efficient consumption of glycerol and promoted the synthesis of reduced products, including ethanol. A previously constructed strain, EH05, containing mutations that prevented the accumulation of competing by-products (i.e. lactate, acetate, and succinate) and overexpressing the fermentative pathway for glycerol utilization [i.e. strain EH05 (pZSKLMgldA)], efficiently converted thin stillage supplemented with only mineral salts to ethanol at yields close to 85% of the theoretical maximum. Ethanol accounted for about 90% (w/w) of the product mixture. These results, along with the comparable performance of strain EH05 (pZSKLMgldA) in 0.5 and 5 l fermenters, indicate a great potential for the adoption of this process by the biofuels industry.
KeywordsBiofuelsMetabolic engineeringGlycerol fermentationEscherichia coliThin stillageEthanol
The commercial production of alternative transportation fuels such as ethanol is an important and growing industry. A variety of feedstocks currently used or under development include corn, sugarcane, sugar beets, and lignocellulosic biomass (Sanchez and Cardona 2008). As the scale of production increases, ethanol producers are faced with the growing challenge of improving the yield of ethanol from the same quantity of feedstock while reducing costs associated with the production process. In a typical ethanol production facility, sugars (e.g. glucose) are converted into ethanol, CO2, glycerol, and small amounts of other chemicals. During the distillation process, the ethanol is removed and further processed according to the manufacturer’s need. The remaining material (often referred to as the column bottoms, vinasse, or whole stillage, depending on the feedstock) may be further processed as a means of recovering value from the by-products (Rausch and Belyea 2006). For ethanol production from corn, the whole stillage is primarily utilized as livestock feed, a route that provides ethanol producers with a substantial revenue source and significantly increases the profitability of the process. However, with the growth of ethanol production in recent years, it is imperative to identify new outlets for these by-product streams in order to maintain the economic viability of this industry (Khanal et al. 2008).
Strains and plasmids
Wild-type E. coli K12 strain MG1655 (F- λ- ilvG- rfb-50 rph-1) was obtained from the University of Wisconsin E. coli Genome Project (www.genome.wisc.edu) (Kang et al. 2004). Strain EH05 is a derivative of MG1655 previously constructed by inactivating fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and lactate dehydrogenase (ΔldhA) (Fig. 1) (Durnin et al. 2009). Plasmids pZSKLMgldA (Yazdani and Gonzalez 2007) and pZSglpKglpD (Durnin et al. 2009) were used to overexpress the fermentative and respiratory pathways, respectively, involved on glycerol utilization (Fig. 1). Manufacturer’s protocols (Qiagen, CA, USA) and standard methods (Miller 1992; Sambrook et al. 1989) were followed for DNA purification, plasmid isolation and electroporation. The strains were kept in 32.5% (v/w) glycerol stocks at −80°C. Plates were prepared using LB medium containing 1.5% (w/v) agar with 34 μg chloramphenicol/ml and 50 μg kanamycin/ml.
Culture medium and cultivation conditions
Culture media were prepared by adding mineral salts to autoclaved thin stillage at the concentrations reported by Neidhardt et al. (1974). MOPS was excluded from the formulation when indicated. Thin stillage generated in a dry grind milling production process was kindly provided by White Energy Holding Company, LLC (Dallas, TX) and had the following characteristics: pH 4.4, Brix 7.6, 8.8% solids, 3.5 g maltose/l, 1.5 g glucose/l, and 24.6 g glycerol/l. Chemicals were obtained from Fisher Scientific (Pittsburgh, PA) and Sigma–Aldrich Co.
Fermentations were conducted in either a 0.5 l (working volume) fermentation system (Ward’s Natural Science, Rochester, NY) or a 5 l (working volume) Biostat A+ from Sartorius Stedim North America Inc. (Bohemia, NY). Both systems have independent control of temperature (37°C), pH (6.3), and stirrer speed.
In the 0.5 l fermenter, a pH controller was fitted using 2 M NaOH for pH control. The stirrer speed was maintained at 200 rpm. The temperature was maintained at 37°C. The dissolved O2 concentration was measured with a DO-BTA dissolved O2 sensor. In the 5 l fermenter, the temperature, pH, agitation (100 rpm) and air flow rate were controlled using manufacturer’s software package (Sartorius Stedim North America Inc., Bohemia, NY).
Microaerobic cultures were established by bubbling air at 1 v.v.m. Operation under these conditions resulted in decreasing dissolved O2 concentrations that fell below the detection limits after 2 h cultivation (i.e., undetectable dissolved O2 concentrations during almost the entire course of the fermentation). The volumetric oxygen transfer coefficient (kLa) under the above operational conditions was 4 h−1 in both fermentors.
Prior to use, the cultures (stored as glycerol stocks at −80°C) were streaked onto LB plates and incubated overnight at 37°C. Ten colonies were used to inoculate 250 ml Pyrex bottles containing 175 ml minimal medium supplemented with 10 g glycerol/l. The bottles were incubated at 37°C and shaken at 150 rpm until an OD600 of ~0.3 was reached. An appropriate volume of this actively growing pre-culture was centrifuged, and the pellet was washed and used to inoculate 350 ml medium in each fermenter, with an initial OD600 of 0.05.
Analytical methods and calculation of fermentation parameters
After centrifugation, supernatants were stored at −20°C for HPLC analysis. Glycerol, sugars, organic acids, and ethanol were measured by HPLC as previously described (Dharmadi et al. 2006; Dharmadi and Gonzalez 2005). The transfer of O2 in microaerobic cultures was characterized by measuring the volumetric O2 transfer coefficient (kLa, h−1) as previously described (Durnin et al. 2009). Data for glycerol consumption and product synthesis were used to calculate average product yields (g product/g glycerol) for 72 h fermentations as previously described (Durnin et al. 2009; Yazdani and Gonzalez 2007).
Results and discussion
Substrate utilization and product synthesis during the fermentation of thin stillage by wild-type E. coli
Effect of overexpression of glycerol-dissimilating pathways on substrate utilization and product synthesis
In E. coli, CCR of catabolic genes is mediated by the combined action of global and operon-specific regulatory mechanisms. The major players in the global pathway are the signal metabolite cAMP, the transcription activator CRP (cAMP receptor protein), the enzyme adenylate cyclase, and the IIA component of the glucose-specific phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) (Goerke and Stulke 2008). Several strategies have been reported to eliminate the sequential and inefficient metabolism of sugar mixtures, all of them based on engineering the aforementioned global regulatory pathways or other components of the PTS (Dien et al. 2002; Nichols et al. 2001; Hernandez-Montalvo et al. 2001). In contrast to previously reported strategies for sugar mixtures, we sought to achieve simultaneous consumption of glycerol and sugars present in thin stillage by circumventing operon-specific regulatory mechanisms: i.e. by directly amplifying the pathways involved on glycerol utilization under microaerobic conditions. These pathways were recently characterized (Durnin et al. 2009) and are shown in Fig. 1.
The overexpression of glycerol-utilization pathways also had a profound effect in the product mixture generated during the fermentation of thin stillage (Table 1). The effect, however, was specific to the pathway being overexpressed. Amplification of the fermentative pathway (gldA and dhaKLM) led to 6- and 10-fold increase in ethanol yield and titer, respectively (Table 1). The relative proportion of ethanol to other fermentation products also increased by about eight times. Interestingly, lactate and succinate production increased as well, while acetate synthesis decreased (Table 1). Overall, reduced products ethanol, lactate, and succinate accounted for 88% (w/w) of the product mixture (i.e. acetate represented only 12%, w/w). These changes are likely due to the fact that glycerol utilization through the gldA–dhaKLM pathway generates reducing equivalents in the form of NADH, which then can be used in the synthesis of ethanol, lactate or succinate (Fig. 1).
Although ethanol production also increased upon overexpression of the respiratory pathway (glpK and glpD), the changes were less pronounced: i.e. 3- and 6-fold increases in ethanol yield and titer, respectively (Table 1). Moreover, the effect on the synthesis of organic acids was opposite to that caused by overexpression of the gldA–dhaKLM pathway: i.e. overexpression of glpK–glpD resulted in higher levels of acetate and lower levels of lactate and succinate (Table 1). Overall, acetate accounted for 40% (w/w) of the product mixture, a 3.4-fold increase over that observed in the case of gldA–dhaKLM overexpression. Taken together, these results indicate that overexpression of the respiratory pathway favors the synthesis of oxidized products (acetate) while overexpression of the fermentative pathway leads to an increased accumulation of reduced products (ethanol, lactate, succinate).
Elimination of byproducts competing with ethanol
Since lactate, acetate, and succinate are major products of the fermentation of thin stillage (Fig. 2; Table 1), we sought to minimize their accumulation by blocking the metabolic pathways responsible for their synthesis. For this purpose, the genes encoding lactate dehydrogenase (ldhA), phosphotransacetylase (pta), and fumarate reductase (frdA), three key enzymes involved in the synthesis of lactate, acetate, and succinate, respectively (Sawers and Clark 2004; Gonzalez et al. 2008; Murarka et al. 2008) (Fig. 1), were disrupted. In the resulting strain, EH05, the synthesis of lactate was completely eliminated and the production of acetate and succinate significantly reduced (Table 1). While maltose and glucose consumption were reduced, glycerol utilization remained at the same low levels observed in the wild type. Although the yield and proportion of ethanol relative to other products increased significantly in strain EH05, the amount accumulated in the fermentation broth was still small (Table 1). The latter suggests that ethanol synthesis may be limited by the poor utilization of glycerol in wild-type and EH05 strains.
Efficient conversion of thin stillage to ethanol
This work was partially supported by a grant from the National Science Foundation (CBET-0645188).