Microbial generation of Bio-ethanol acting as an alternative to the exhaustive forms of fuel

Microbial generation of Bio-ethanol acting as an alternative to the exhaustive forms of fuel

A worldwide interest in the utilization of bio-ethanol as an energy source has stimulated studies on the cost and efficiency of industrial processes for ethanol production. Intense research has been carried out for obtaining efficient fermentative organisms, low-cost fermentation substrates, and optimum environmental conditions for fermentation to occur. Traditionally, ethanol has been produced in batch fermentation with yeast strains that can- not tolerate high concentration of ethanol. This necessitated the strain improvement programme for obtaining alcohol-tolerant strains for fermentation process.Zymomonas mobilis, a gram-negative bacterium, is considered as an alternative organism in large-scale fuel ethanol production. Comparative laboratory- and pilot-scale studies on kinetics of batch fermentation ofZ.mobilis versus a variety of yeast have indicated the suitability ofZ. mobilis over yeasts due to the following advantages:

i. its higher sugar uptake and ethanol yield,

ii. its lower biomass production,

iii. its higher ethanol tolerance,

iv. it does not require controlled addition of oxygen during the fermentation, and

v. its amenability to genetic manipulations.

Due to dwindling of fossil fuel, microbial production of bio-fuel from organic byproducts has acquired significance in recent years. Ethanol has been trusted as an alternate fuel for the future. Even though several microorganisms, includingClostridium sp., have been considered as ethanologenic microbes, the yeastSaccharomyces cerevisiae and facultative bacteriumZymomonas mobilis are better candidates for industrial alcohol production.Z. mobilis possesses advantages overS. cerevisiae with respect to ethanol productivity and tolerance, thus encouraging researchers for exploitingZ. mobilis ability to utilize sucrose, glucose, and fructose by EntnerDeudoroff pathway. The bottlenecks inZ. mobilis are: (i) its inability to convert complex carbohydrate polymers like cellulose, hemicellulose, and starch to ethanol, (ii) its resulting in byproducts such as sorbitol, acetoin, glycerol, and acetic acid, and (iii) formation of extracellular levan polymer. To circumvent these problems, genetic manipulation ofZ. mobilis has been attempted for broadening the utilizable range ofZ. mobilis, i.e. genes encoding several hydrolytic enzymes from related bacterial species have been cloned, and transferred intoZ. mobilis. Interestingly, apet operon (production ofethanol) was constructed by combiningpdc (pyruvate decarboxylase) andadhII (alcohol dehydrogenase) genes ofZ. mobilis, and transferred to other bacterial strains to make them ethanologenic novel strains. Through classical mutation and selection approaches, mutants ofZ. mobiliswith improved fermentation characteristics and without byproduct formation have been obtained. In addition to ethanol,Z. mobilis has also been metabolically engineered to produce L-alanine and L-lactic acid. Genes encoding b -carotene synthesis have also been cloned and successfully expressed inZ. mobilis to enrich the fermented nutrients of farm animals. Several applications of levan in food and pharmaceutical industries provide an opportunity to exploitZ. mobilis for large-scale production of levan. The merits ofZ. mobilis suggest the potential use of this organism in industrial production of various fermentation products.

The only limitation ofZ. mobilis compared to the yeast is that its utilizable substrate range is restricted to glucose, fructose, and sucrose.Z. mobilis was originally isolated from alcoholic beverages like the African palm wine, the Mexican pulque', and also as a contaminant of cider and beer in European countries. On the basis of evaluation using the modern taxonomic approaches, the genusZymomonas has only one species with two subspecies,Z. mobilis subsp.mobilis andZ. mobilis subsp.pomaceae. The ability to utilize sucrose as a carbon source distinguishesZ. mobilis fromZ. anaerobia. It is one of the few facultative anaerobic bacteria which metabolizes glucose and fructose via the EntnerDeudoroff (ED) pathway, which is usually present in aerobic microorganisms. Under anaerobic conditions,Z. mobilis produces byproducts such as acetoin, glycerol, acetate, and lactate, which result in reduced production of ethanol from glucose. During growth ofZ. mobilis in fructose, the formation of ace-toin, acetic acid, and acetaldehyde was clearly more pronounced than when grown in glucose. However the cell yield was low during its growth in fructose.In addition to ethanol fermentation,Z. mobilis has potential application in polymer production. Levan, a polymer of fructose units linked by b -2,6-fructosyl bond, is produced byZ. mobilis during its growth on sucrose medium. Microbial levan is of commercial importance and is used as a thickening, gelling, and suspending agent. In recent years, strategies to improve the yield of levan production by microorganisms attracted greater attention.

Ethanol production byZ. mobilis has been restricted to glucose, fructose, and sucrose substrates. Alternatively, crude sucrose substrates such as sugar beet, bagasse and molasses have also shown promise as substrates for direct fermentation to ethanol. Recombinant DNA technology could be exploited to construct microbial strains to produce ethanol from these low-cost agricultural substrates. To obtain ethanologenic strains utilizing the above- mentioned substrates, eitherZ. mobilis was transformed with genes of interest acquired from other organisms or gene ofZ. mobilis involved in ethanol synthesis was transferred to other organisms with required characteristics.

Continuous fermentation

Among various kinds of fermentation processes studied, a continuous process using co-immobilized AMG and cell was most favorable, with operational stability for over 40 days. Continuous production of ethanol from Jerusalem artichoke Juice using ZM4F ofZ. mobilis was studied by Allaiset al. Their results showed the volumetric productivity to be 67.2g/l/h with a final ethanol concentration of 42g/l from 100g/l initial sugars. Doelledescribed a process for the continuous production of ethanol from hydrolysates of starch. This process made use ofZ. mobilis in a single-stage fermentation. The author maintained that the quality of the starch hydrolysate was not crucial to the success of the fermentation, and reported a conversion efficiency of 92%. A process for the continuous production of ethanol on an industrial scale from hydrolysed wheat starch usingZ. mobilis was described by Sahm and Bringer-Meyer. These investigators reported that a strain ofZ. mobilis that produced 60g ethanol/l over a test period of 39 days was used for the industrial-scale fermentation. Hillaryet al. studied continuous culture

using single- and double- fermentor systems, and reported that this system had higher productivity than immobilized enzyme systems. Biofilm reactors with polypropylene or plastic support were used for ethanol production. Results showed that the ethanol production rate and concentration were greater in biofilm reactors than in suspension cultures. In continuous fermentation using mixed cultures of Z. mobilis and S. cerevisiae, production of 54.3g/l of ethanol was observed within 3 days. These authors reported that a high ethanol productivity of 70.7g/l/h was obtained with a final ethanol concentration of 49.5g/l and yield of 0.5g/g. This amounted to 98% of the theoretical yield and 99% substrate conversion. Therefore this might be considered as right candidate for increasing the rate of the ethanol production in the existing industries.

Solid state fermentation:

In recent years, considerable interest has been shown in using agricultural byproducts such as sweet sorghum, corn, apple, grape, sugar cane, sugar beets, fodder beets, and Jerusalem artichoke tubers for fuel ethanol production. Due to the complex composition and insolubility of these agro-substrates, solid-state fermentation of these sources would be economical. Very few reports are available regarding the production of ethanol by solid state fermentation. Ethanol fermentation in solid state byZ. mobilis grown on sugar-beet shown a yield of 0.48g/g sugar, volumetric productivity of 12g/l/h and final ethanol concentration of 130g/l showed good performance ofZ. mobilis in a solid-state fermentation. Furthermore, it has beenreported that during solid-state fermentation fewer by-products were produced, compared to conventional submerged fermentation. At optimum fermentation temperature of 35C, an ethanol yield of up to 95% of the theoretical value with final ethanol concentration of 142g/l were obtained.

Conclusion

In recent years, attention has been focussed on effective utilization of agro-byproducts to produce fuel usingZymomonas mobilis. A thorough investigation of molecular biology and biochemistry of ethanol production byZ. mobilis has been accomplished.Owing to the genetic amenability ofZ. mobilis, it is possible to make use of this organism in industrial production of ethanol. Thus, it is time that the industrialists collaborate with academicians to translate the laboratory findings in science for the benefit of society.

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