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Depletion of natural resources of fossil fuel makes it a necessity to look for an alternative source of energy. In this direction the first and most suitable option we can find out is of Ethanol. Ethanol is a sustainable source of energy which is now one the most important need for the time. Also environmental issues are better tackled with ethanol as compared to fossil fuels. It emits lower greenhouse gases and hence can lower the carbon output from its combustion. The increased political pressure from countries like United States and European countries for energy security to reduce oil dependency on other countries has supported use of ethanol as fuel and they look towards an ethanol as future alternative to meet energy need. (KEVIN A GRAY, LISHAN ZHAO and MARK EMPTAGE, 2006). Also its higher octane number, higher heat of vaporisation and compatibility with engines with few modifications has allowed its use as blend with petroleum product but even use of 100% ethanol as a fuel is the ultimate aim. Countries like Brazil and United States are the world leaders to promote use of ethanol. There are many uses of ethanol apart from its use as fuel but it is the use of ethanol as renewable source of energy to meet the need of country's transportation and energy need for the economic growth that has prompted the biological production of ethanol. (B. HAHN-HA¨ GERDAL, et al., 2006)
Although ethanol can be produced with chemical methods, the biological methods are of more interest as far as use of ethanol as fuel is concerned. It is because it uses the renewable resources and recycles the biomass already available. The biological approach makes use of different carbon sources ranging from simple sugars like glucose, fructose etc. to complex polymers like starch, cellulose etc.
Zymomonas mobilis - Mysterious ethanologen
For years and even today Saccharomyces cerevisiae is the major organism involved with alcohol production. But the ethanol mainly they produce are used as beverage. But recent need for much higher amount of ethanol need has proven that we need to look for organism with superior properties producing higher ethanol yield. This has prompted us to look back in time and hence Zymomonas mobilis has regained interest as an alternative alcohol producing organism.
Zymomonas mobilis was originally isolated as a contaminant from cider, bear. Also it was isolated from maxican "pulque" and African wine. This created the interest in scientists to isolate and study the contaminant to prevent the loss of the alcohol industry caused due to organism. (JACQUES C. BARATTI and J. D. BU'LOCK, 1986)
It is a gram negative facultative anaerobic organism. The most important characteristic of Zymomonas mobilis is its ability to degrade glucose by Entner- Doudoroff pathway, this makes organism unique as an ethanol producer.
Not only ethanol but Zymomonas mobilis is capable of producing many high value products like levan polymer, carotenoids, but for our discussion will focus entirely on ethanol production ability of the organism.
Why Zymomonas mobilis?
Zymomonas shows many unique characteristics that make it superior over conventionally used yeast for alcohol production such as:
Zymomonas has higher sugar uptake rate as is mediated by facilitated diffusion. It gives higher ethanol yield as it utilises less carbon to generate ATP but most of the substrate is converted into ethanol. It produces lower growth rate due to less ATP production and hence produces higher ethanol per unit media component. It does not need addition of oxygen at controlled rate. It allows easier genetic manipulation for industrial strain improvement. It produces lesser quantities of side products like glycerol, acetate, succinate, acetoin etc which results in increased alcohol yield with easier purification steps. (PARMJIT S. PANESAR, SATWINDER S. MARWAHA and JOHN F. KENNEDY, 2006)
Only limitation of the organism is the range of substrates it can use but now with advancement of technology like genetic engineering and fermentation technologies these limitations can be overcome.
Taxonomy, Molecular biology & Biochemistry of Zymomonas mobilis
Naturally occurring Zymomonas can ferment glucose and fructose directly to produce ethanol. The problem arises with the utilisation of sucrose as it produces leven which is a polymer of fructose or sorbitol and hence various approaches have been used to exploit the ability of Zymomonas to produce ethanol from sucrose.
To date all the strains identified belongs to the single species Z. mobilis with two known subspecies Z.mobilis subsps. mobilis- important in industrial ethanol production and Z.mobilis subsp. pomaceae- responsible for beer and cider spoilage.
Z. mobilis is resistant to many of the commonly used antibiotics. The genome of the Z.mobilis is about 1.5Ã-109 bp and possesses about 1500 cistrons. The GC- content is about 47.5%. (P.L.ROGERS, Y.J.JEON, K.J.L. and H.G.LAWFORD, 2007)
The structural characteristic of Zymomonas mobilis is that makes more tolerant to higher ethanol concentration. This is correlated to the membrane structure of the Zymomonas mobilis. It contains reduced lipid to protein content in the membrane in response to ethanol. But the noteworthy component of the membrane is the presence of cis-vaccenic acid constituting more than 70% of the membrane fatty acid. Also higher quantity of hopanoids present in the membrane in higher quantity aid in tolerating higher ethanol concentration. Also entry of glucose and fructose in the cell is by facilitated diffusion. Because of this even under higher glucose concentration cell is able to survive as it balances higher extra cellular osmotic pressure with rapid uptake of sugar increasing intracellular concentration. (STEVEN E. BUCHHOLZ, MARGARET M. DOOLEY and DOUGLAS E. EVELEIGH, 1987).
Zymomonas utilises Entner Doudoroff pathway to produce pyruvate from sugar. This is an alternate pathway to glycolytic pathway. It produces only 1 ATP per molecule of sugar used. Also it produces 1 NADH and 1 NADPH. In very few organisms this is an alternate pathway used to produce pyruvate. The enzymes catalysing the key limiting steps are pyruvate decarboxylase and alcohol dehydrogenase. The enzyme alcohol dehydrogenase is unique and critical as it continues fermentation even under higher concentration of ethanol. The enzyme machinery of the organism is tolerant to very high ethanol concentration and as a result even cell free system rapidly produces higher ethanol concentration. (P. GUNASEKARAN and K. CHANDRA RAJ, 1999). This feature of enzyme machinery together with unique membrane constituents makes Zymomonas mobilis more tolerant to ethanol.
Growth Conditions for Zymomonas mobilis
The growth requirement of Zymomonas mobilis is very stringent and that is the reason that even though it is known for many years but there are limited defined synthetic and semi synthetic media where it can grow. As far as carbon source is concerned as mentioned it can utilise only glucose, fructose and sucrose. Only few of the genetically modified strains have been developed which can utilise some of the polysaccharides and pentose as carbon source for growth and alcohol production as will be discussed later. It was thought that the organism needs organic nitrogen only for growth like tryptone, but in some of the media addition of only (NH4)2SO4 allows organism to grow. Apart from this it is important to include metal ions like magnesium, manganese, iron etc. The importance of addition of growth factors is also explored and hence yeast extract is often included while designing a media. The growth will not take place until addition of vitamins which differs as per the strain that is being cultivated. But the vitamins being identified as important are nicotinic acid, calcium pentothenate, pyridoxine hydrochloride, biotin and thiamine. (ARNOLD L. DOMAIN and NADINE A. SOLOMON, 1985), (S. SIVA KESAVA, S. K. RAKSHIT and T. PANDA, 1995)
Zymomonas mobilis can grow over wide pH range of 3.0-7.0 but optimum being 5.0-6.5. Also most of the Zymomonas strains will tolerate 1.0% NaCl but members of the subspecies pomaceae are unable to grow even at a standard NaCl concentration of 0.5%-0.7%. Optimum temperature range was found to be 300 C -400C but from industrially point of view the yield of the process decreases after 350C but the optimum temperature for maximum product yield depends upon the carbon source used for the process. (M.L. CAZETTA, et al., 2007)
Strain Improvement of Zymomonas mobilis
Various approaches have been tried to produce ethanol from substrates using Batch and Continuous cultures. The results have been promising and that is the reason the potential of Zymomonas mobilis as superior ethanol producer have been identified. But due to limitation of the cheap substrate utilisation organism could not receive industrial importance and in order to exploit the characteristics of organism scientist used approaches to overcome these limitations.
There are two major approaches to broaden the substrates that the organism can utilise. The approaches are:
Genetic engineering approaches
Novel approaches: Co-culture technique and Simultaneous Saccharification and Fermentation (SSF)
Genetic Engineering approaches
Genetic engineering of either Z.mobilis with genes from other organisms or transfer of genes from Z.mobilis to other organisms are the approaches used to obtain strain that can tolerate higher sugar concentration and can yield higher ethanol concentration. (GEORG A. SPRENGER, 1993). This approach was developed in accordance with the substrate utilisation by Z.mobilis that was desired. The waste stream from industries like dairy, fruit processing and paper industry had influenced the development of recombinant Zymomonas strains.
Lactose utilisation was one of the characteristic desired to be present in Z.mobilis so that dairy waste enriched in lactose can be efficiently converted into ethanol. Hence the lac operon from E.coli was transferred to Z.mobilis using variety of vectors like plasmids, transposons with important genes fused but due to problems like low expression of genes, lower permease activity and slow growth limited the success of the approach. Similar results were obtained from recombinant strain created to use raffinose.
Many times plant wastes are used for ethanol production. Plant cell walls contain hemicelluloses, lignocelluloses (J. P. DELGENES, R. MOLETTA and J. M. NAVARRO, 1996) rich in xylose which is known to be transported into the cells of Z.mobilis. Hence two enzymes were needed to be present xylulokinase and xylose isomerase and many attempts were made to introduce these enzymes in the Z.mobilis so that xylose can be converted to intermediate of Entner- Doudoroff pathway and the approach was successful. The engineered strain of Z.mobilis grew on xylose. (B.S. DIEN and M.A.COTTA, T.W.J., 2003) Apart from this two other genes were also transferred from E.coli to Z.mobilis on a plasmid so that xylose can be fermented efficiently. Similarly five genes were transferred from E.coli through a plasmid to utilize arabinose as substrate for conversion to ethanol. Also resulting strain shows much higher ethanol production in xylose/glucose mixtures. (CHUN-QIANG LIU, AMANDA E. GOODMAN and NOEL W. DUNN, 1988)
It is always desired characteristic to utilise polysaccharides as substrate for conversion to ethanol due to easy, abundant and cheap availability. Hence attempts have been made to engineer strains of Zymomonas mobilis to utilise starch and cellulose without pre-treatment directly to ethanol. It would be consist of two steps where in the first step polysaccharides would be converted to simpler monosaccharide which can then be converted to ethanol.
To utilise starch as a substrate Î±- amylase was the enzyme, gene for which was cloned from Bacillus licheniformis. Secretion of the enzyme was confirmed but utilisation of starch could not be confirmed for growth. Also after few generations the plasmid cloned in Z.mobilis carrying gene for Î±- amylase was lost as reported by GEORG A. SPRENGER, 1993. But recent studies have successfully created recombinant Z. mobilis starins that is able to carry out fermentation using cassava starch, sago starch, wheat stillage and potato starch as substrate to produce ethanol and with excellent conversion efficiency as well as ethanol yield. (LINDA DAVIS, et al., 2006)
Gene celz encoding endo-Î²-glucanase was cloned from Erwinia chrysanthemi which makes an initial attack on cellulose was cloned in Z.mobilis and which resulted in expression of genes (NADINE BRESTIC-GOACHET, et al., 1989). Another gene was cloned in Z.mobilis to utilise product of the cellulose that is disaccharide cellobiose. The enzyme responsible for utilisation of cellobiose is Î²-glucosidase which was cloned from Xanthomonas albilineans and in another approach from Rurninococctt albus in Z.mobilis and as a result successful production of ethanol was confirmed but the yield was very low, about only 30% of the theoretical yield. The problem here was associated with the uptake of cellobiose and it created a decrease in the ethanol yield. Also using recombinant plasmid pNB20 along with helper plasmid RP4 cellulase enzyme coding genes were transferred to Zymomonas mobilis providing with a characteristic to produce cellulose degrading enzyme. It was proved that it was expressing the enzyme coding genes resulting in the release of the about 35% of the enzyme into the growth medium. These results are indicating that foreign genes coding for the complex enzyme can be expressed in Zymomonas mobilis and there is possibility that if suitable conditions are provided then substrate utilization range can be widened making organism suitable for the industrial fermentation.
Gene Transfer from Zymomonas mobilis to other organisms
In another approaches genes from Zymomonas mobilis are being transferred to the other organisms already used in industry to produce microbial products but their characteristics can be improved to make them more osmo-tolerant and alcohol tolerant, so that they can be used to produce ethanol, e.g. To utilise dairy waste rich in lactose and expensive to treat for disposal. It is more economical to use this whey as substrate for alcohol production. So to divert the pathway that converts pyruvate to lactate, Lactic acid producing bacteria were engineered to convert pyruvate to ethanol (NANCY N. NICHOLS, BRUCE S. DIEN and RODNEY J. BOTHAST, 2003). For these two genes were transferred to lactic acid producing bacteria using plasmid vectors. These genes were pyruvate dehydrogenase and alcohol dehydrogenase from Zymomonas mobilis. Although the lactic acid was still a major primary product and very low levels of alcohol was produced but the research is now being focussed in this aspect as well and many bacteria are trying to be incorporated with genes from Zymomonas mobilis. Some of these are Klebsiella oxytoca, organism capable of degrading cellulose etc.
Novel Approaches to improve ethanol production by Zymomonas mobilis.
Pre-treatment of complex substrates with mineral acids or enzymes to convert it into monomers was one of the approaches for batch production of ethanol but inhibitory end products often caused death of fermenting organism, lower yield of ethanol or contamination by inhibitory products.
One of the approaches used now, to improve the range of substrates that can be utilised by Z.mobilis, is the simultaneous saccharification and fermentation. Here Z.mobilis is cultured with any organism possessing the property of producing an enzyme that can convert a polymer into saccharified (simpler) product which can be utilised by Z.mobilis to convert into ethanol. There are many examples of using this approach successfully to produce ethanol with higher yield. The approach was used to study the use of cassava starch by Z.mobilis to produce ethanol. The other culture used here was Saccharomyces uvarum. (P. GUNASEKARAN and K. CHANDRA RAJ, 1999) the yield for the process was found to be much higher as compared to individual activities and approximately 95% of theoretical yield was obtained in much lesser time. The success of the approach depends upon factors like state and purity of the substrate. In case of cultures Z.mobilis and Saccharomyces fibuligera higher ethanol yield were obtained by Simultaneous Saccharification and Fermentation but amount of product could have been higher with liquefied starch. Many other approaches have been tried to use in conjunction with simultaneous saccharification and fermentation like immobilisation of cultures, use of immobilised enzyme and Z.mobilis or immobilisation of Z.mobilis and use of enzyme.
There are many novel approaches that are being experimented to utilise biomass available to produce ethanol. Many combinations of approaches, where genetic engineering along with Simultaneous Saccharification fermentation (SSF) are implemented, have been tried out. Even in these approaches Zymomonas mobilis is the key player. One of the examples of this approach is presented by HELEN GOLIAS, et al., 2002. In the research carried out. Recombinant was able to produce considerable ethanol in lesser period but due to lesser ethanol tolerance it was producing lower yield. When the approach involved co-culturing with Zymomonas mobilis, co-culture was able to produce higher ethanol yield at very high rate. Similar approach of co-culture was utilised by co-culturing Kluyveromyces fragilis and Zymomonas mobilis to produce ethanol from lactose (NUMBI RAMUDU KAMINI and PARAMASAMY GUNASEKARAN, 1989).
Different approaches have been tried not only with the genetically engineered organisms and the variety of substrates used but also with the other process parameters. One of the approaches utilizes the property of acid tolerant mutant of Zymomonas mobilis. In the process fermentation was carried out under non-sterile conditions as the mutant was capable of starting fermentation at pH 4.5 which does not allow many organisms to grow and hence maintain sterile condition. The process ethanol yield was higher due to more available glucose then is available after autoclaving. Also it reduces the energy consumption by about 40% that is required to maintain sterile condition. Although these process has some constraints like presence of contaminants even though they do not grow, but modified processes like SSF to limit the amount of glucose available for use by contaminants, include antibiotics can overcome the limitations. (F. TAO, et al., 2005)
Research is currently being done to produce ethanol from any source of biomass whether it is from dairy industry rich in lactose, fruit processing industry producing orange peel oil (MARK R. WILKINS, 2009), pineapple cannery waste (J.N. NIGAM, 2000), food industry could be rich in starch like wheat stillage (LINDA DAVIS, et al., 2005), natural rubber waste (SUDARUT TRIPETCHKUL, MICHIO TONOKAWA and AYAAKI ISHIZAKI, 1992), paper sludge waste from paper and wood-pulping industry (YUYA YAMASHITA, et al., 2008). This has prompted to carry out more research with Zymomonas mobilis to broaden its substrate utilization range.
Advancement in technology has provided a novel technique to carry out industrial biotechnology processes efficiently. The latest advancement is Immobilization technique. This has been experimented in many directions ranging from immobilization of single cell to co-cultures and enzymes. In all approaches yield of ethanol was improved then free cell cultures. In a recent experiments by M. REBROSË‡, et al., 2005. Zymomonas mobilis cells were immobilised in polyvinylalcohol (PVA) matrix. This improved both batch and continuous production of ethanol. The benefits of the approach were long term stability of the process as the cultures can be reused for many cycles, also high volume of productivity as it increased ethanol tolerance of the organism. There were many other technological advantages that it has provided.
In another approach cells of Z.mobilis were immobilized with amyloglucosidase for producing ethanol from sago starch (C. H. KIM, et al., 1988). In this Simultaneous Saccharification fermentation (SSF) process cells were immobilised in Sodium alginate and Îº-carrageen matrix. The process was quite successful with ethanol yield equal to 93% of theoretical yield.
Immobilisation studies have also been carried out to study co-culture of yeast and bacteria for conversion of glucose/xylose mixture to ethanol. In the experiment yeast Pichia stipitis and bacteria Zymomonas mobilis were studied. The yeast cells were free in the fermentation process and Z.mobilis cells were immobilised. The ethanol yield obtained was more than 96% of the theoretical yield and also utilization of substrates was improved. The approach used was successful in overcoming the inhibition of P.stipitis from utilising xylose due to oxygen consumption by Z. mobilis and higher ethanol yield was obtained. (NAN FU, et al., 2009). Even addition of immobilised Sucrase improved ethanol production by Zymomonas mobilis (WEN-CHIEN LEE and CHENG-TA HUANG, 1995)
Increasing political pressures, rising oil prices and awareness about environmental issues are demanding fuels from renewable feedstocks are acting as catalyst for studies on Zymomonas mobilis. In spite of great advantages offered by the organism that makes it a potential solution to meet the requirement of global ethanol need, there is no commercial institution currently exists carrying out fermentation using Zymomonas mobilis to produce ethanol. In this assignment I have tried to cover most of the dimensions in which research is directed that can help in exploiting the characteristics of Zymomonas mobilis to meet industrial demands whether it is wider substrate utilisation by the organism or more efficient fermentation process producing higher ethanol yield. The approaches like strain improvement, SSF and immobilisation technology will certainly provide us the opportunity to identify potential exploitation ability of Z. mobilis that can make it a preferred organism producing ethanol in near future.