2.3. Bioethanol fermentation process and methods to improve this process
2.3.2. Methods (techniques) to improve bioethanol fermentation
One of the improvements for ethanol generation by biological routes is the invention of new methods for fermentation, as well as downstream separation approaches. Fermentation coupled with prevaporation was carried out by O’Brien and Craig [136], however, by assessing the economics of this technique with typical dry-milling ethanol fermentation, it was found that this technique was more expensive. Another invention was the use of vacuum fermentation for ethanol generation to decrease ethanol concentration in fermentors [137, 138]. However, this technique is impractical for fermentors working with huge volumes of broth (hundreds to thousands of cubic meters), since controlling vacuum conditions in these bioreactors requires significant costs, not only for designing instruments, but also in their operation. This led to this technique having a low impact in the fermentation industry. A pilot plant operation where ethanol fermentation was combined with a stripping column and a condenser was introduced by Tylor et al. [139]. In this system, ethanol was stripped by the recycled CO2 in the stripping column and the ethanol enriched CO2 entered a low temperature condenser in which ethanol was absorbed by the circulated dilute ethanol condenser [139]. However, the disadvantage of this system is the complexities in both its design and operation, leading to poor economics.
Another aspect aimed at improving ethanol fermentation was the development of bioreactors to decrease ethanol inhibition by reducing fermentor backmixing, for example the use of batch bioreactors and plug flow bioreactors are the best selections since there is no backmixing inside these types of bioreactors [140]. However, the disadvantage of the batch fermentation is long periods of operational downstream, time required for mash addition, broth harvesting, tanks and pipeline cleaning, meaning it is unsuitable for large scale fermentation [140]. The use of biofilm reactors to produce ethanol have also been developed to improve the economics and the performance of the fermentation process [141]. Additionally, the immobilized cells technique has been also investigated to eliminate inhibition caused by high concentration of both substrate and product, allowing enhanced productivity and yield [142-145]. Recently, ethanol fermentation using an immobilized cell reaction was successfully demonstrated to improve ethanol production [146]. However, for VHG fermentation, the inhibition of both substrates (sugar) and products (ethanol) will be considered, and therefore, methods to improve the fermentation process will be also considered.
2.3.2.2. Improvement of media quality
In the terms of “enhanced ethanol concentration”, many studies [147-155] have been carried out to achieve higher ethanol concentration in the broth. For industrial ethanol fermentation, sugar concentrations in the broth above 200 g/L are not applied since the high concentrations of substrates and ethanol (formed from fermentation) lead to delay in the growth of yeast, slowing down the fermentation [156]. Therefore, many attempts to improve the media quality for ethanol fermentation have been carried out, including the addition of supplements such as, calcium [147], magnesium [148, 149], ammonium [150, 151], glycine [152], peptone [153] and yeast extract [154, 155], since these factors play protective effects either on growth, fermentation, or viability of cells, which can stimulate the rate of ethanol production. By modifying the nutritional conditions, ethanol production in a fed-batch process with S. cerevisiae was increased to 85.8 g/L in 125 h [157].
Moreover, a higher ethanol tolerant yeast strain was used to produce up to 103.7 g/L of ethanol in 66 h [158]. Thomas et al. [155] introduced a process called very high gravity (VHG) conditions using the bioconversion of hydrolyzed wheat supplemented with yeast extract for ethanol fermentation that can help to produce up to 150 g/L in 175 h [155].
Recently, Alfenore et al. [159] also succeeded in enhancing ethanol fermentation by a vitamin feeding strategy (including addition of pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, and biotin) during a fed-batch process, enabling the biosynthesis of 147 g/L of ethanol [159].
2.3.2.3. Metabolic engineering
With respect to improving ethanol fermentation in the terms of an “optimization of fermentation process”, many studies have been carried out. As detailed above, the formation of ethanol is redox-neutral, but other cellular processes also produce NADH, which is reoxidized by the glycerol-3-phosphate dehydrogenase (Gpd1p) to avoid an imbalance in the NAD+/NADH ratio. One of the ways to decrease excess NADH in yeast is the formation of glutamate, which relates to two coupled reactions, catalysed by the NADH-dependent glutamate synthase (Glt1p) and glutamine synthetase (Gln1p) [160].
Therefore, overexpression of the GLT1 gene could increase the conversion of NADH to NAD+. This may also decrease the role of Gpd1p in the NADH reoxidation reaction, leading to lower glycerol production [160]. The formation of glycerol will consume up to 5% of the available carbon, therefore, decreasing glycerol formation can increase the efficiency of ethanol production. Based on this, Nissen et al. [160] deleted the GDH1 gene and overexpressed the GLN1 gene to create the mutant strain TN19. This resulted in a 10%
higher ethanol yield and a 38% lower glycerol yield, compared to the wild type. Similarly, Kong et al. [161, 162] improved ethanol fermentation by deleting the FPS1 gene (whose function is to mediate diffusion of glycerol to the extracellular environment [163]) and overexpressed GLT1 in S. cervisiae [161], as well as overexpressed GLT1 in a gpd2U
mutant strain [162]. This resulted in 13-14% higher ethanol production and 30-38% lower glycerol formation compared to wild type [161, 162].
In terms of “improving cell viability” for ethanol fermentation, Alper et al. [164] used a technique called global transcription machinery engineering for reprogramming gene transcription to create a mutant strain that showed enhanced cell viability for ethanol generation [164]. Moreover, in a separate study, the overexpression of LEU1 also enhanced cell viability for ethanol fermentation [165].
In brief, many attempts (as discussed above) have been made to improve ethanol fermentation, however, a “perfect system” for ethanol fermentation is yet to be created.
Indeed, the inventions of new technical processes increase the investment (capital) of plant;
or the improvements of media quality can increase ethanol concentration. However, increases of by products are also observed. Metabolic engineering techniques can help to eliminate by product biosynthesis, however, high ethanol concentration in the broth can not be observed. Therefore, “a synchronized system” established via the application of the right yeast strain, and ideal fermentator for ethanol production, is required to achieve enhanced ethanol biosynthesis, reduced by product synthesis and improved economics of the fermentation process.